Targeting of ews-fli1 as anti-tumor therapy

ABSTRACT

Peptides and compounds are provided that function as EWS-FLI1 protein inhibitors. The peptides and compounds have utility in the treatment of Ewing&#39;s sarcoma family of tumors. Also provided are methods of preparing the compounds and assays for identifying inhibitors of EWS-FLI1 protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/494,191 filed Jun. 29, 2009. U.S. application Ser. No. 12/494,191 isa continuation-in-part of PCT International Application No.PCT/US2007/089118 filed Dec. 28, 2007 under the Patent CooperationTreaty (PCT), which was published by the International Bureau inEnglish, which designates the United States and claims the benefit ofU.S. Provisional Application No. 60/877,856 filed Dec. 29, 2006. U.S.application Ser. No. 12/494,191 claims the benefit of U.S. ProvisionalApplication No. 61/177,932 filed May 13, 2009. The disclosures of eachof the foregoing applications are hereby expressly incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under NIH Grant/ContractNumbers R01CA138212 and R01CA133662 awarded by the National Institutesof Health of the United States of America. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledGTWN_(—)010CP1C1.TXT, created Mar. 9, 2010, which is approximately 9 KBin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Peptides and compounds are provided that function as EWS-FLI1 proteininhibitors. The peptides and compounds have utility in the treatment ofcancers including the Ewing's sarcoma family of tumors, pancreaticcancer, prostate cancer, and other cancers comprising translocation genefusions. Also provided are methods of preparing the compounds and assaysfor identifying inhibitors of EWS-FLI1 protein.

BACKGROUND OF THE INVENTION

EWS-FLI1 has been identified as a critical target in Ewing's SarcomaFamily of Tumors (ESFT) over 15 years ago, yet no therapies haveheretofore moved from bench to bedside that have impacted on the outcomeof the disease. While many investigators have recognized the importanceof this target, the biochemical nature of EWS-FLI1 presentsdrug-discovery challenges.

The paradigm of disrupting key protein interactions may have utility intreatment of other diseases including sarcomas (Helman L J, Meltzer P.Mechanisms of sarcoma development. Nat Rev Cancer 2003; 3(9):685-94)with similar translocations, and leukemias with MLL translocations (PuiC H, Relling M V, Downing J R. Acute lymphoblastic leukemia. N Engl JMed 2004; 350(15):1535-48). A recent review suggests that disorderedproteins may be excellent therapeutic targets based on their intrinsicbiochemical properties (Cheng Y, LeGall T, Oldfield C J, et al. Rationaldrug design via intrinsically disordered protein. Trends Biotechnol2006; 24(10):435-42).

Despite years of in vitro and xenograft studies with antisense and siRNAdirected towards EWS-FLI1, none of these is heretofore practical as ahuman therapy based on inadequate delivery and stability. A recent phaseII clinical trial using Ara-C was begun in patients with ESFT based on acomparison of cDNA signatures between siRNA reduced EWS-FLI1 and a panelof FDA approved compounds in an ESFT cell line. The recognition thatAra-C may become useful in ESFT therapy is important, however, there aremany reasons not to rely on this early result and to pursue morespecifically targeted therapy. Ara-C has a broad spectrum of activitythat is very dose-dependent and while it may demonstrate activity forESFT patients, its mechanism of action is more generalized than simplyinactivating EWS-FLI1 with broader side effects as well. Ara-C does notrepresent the kind of specifically targeted therapy that might result ina major breakthrough for ESFT patients (both to improve survival andreduce long term effects of therapy).

SUMMARY OF THE INVENTION

A specific and targeted medicine to inhibit a disordered protein,functioning as a transcription factor, without enzymatic activity isdesirable. Therapeutic compounds and novel chemical probes to modulateEWS-FLI1 function are provided that tightly and specifically bind toEWS-FLI1 are also desirable.

Accordingly, in a first aspect, a peptide is provided comprising asequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 and 31, preferably SEQ ID NO: 29.

In an embodiment of the first aspect, the peptide further comprises anN-terminal tag comprising a cell-penetrating cationic peptide. Thecell-penetrating cationic peptide may comprise the cell-permeableAntennapedia peptide sequence (SEQ ID NO: 34).

In a second aspect the peptide in combination with at least onepharmaceutically acceptable carrier or diluent is provided.

In a third aspect, a method for treating cancer in a mammal, comprisingadministering to the mammal an effective amount of the peptide of thefirst aspect is provided.

In an embodiment of the third aspect, the cancer comprises atranslocation gene fusion.

In an embodiment of the third aspect, the cancer is selected from thegroup consisting of Ewing's sarcoma, clear-cell sarcoma, myxoidliposarcoma, desmoplastic small round-cell tumor, myxoid chondrosarcoma,acute myeloid leukemia, congenital fibrosarcoma, prostate cancer andpancreatic cancer.

In an embodiment of the third aspect, the cancer is Ewing's sarcoma.

In an embodiment of the third aspect, the peptide of the first aspect isadministered in combination with another pharmaceutically active agent,either simultaneously, or in sequence.

In a fourth aspect, a use of a peptide of the first aspect for themanufacture of a medicament for the treatment of cancer is provided.

In a fifth aspect, a compound is provided having a formula:

wherein R₁ is selected from the group consisting of hydrogen, one aminoacid, two amino acids linked together, three amino acids linkedtogether,

R₆ is C₁₋₆ dialkyl amine; R₇ is selected from the group consisting ofhydrogen and C₁₋₆ alkyl;R₈ is C₁₋₆ alkyl; R₃, R₄, R₅, R₉, R₁₀, R₁₁, R₁₂, R₁₃, and R₁₄ areindependently selected from the group consisting of hydrogen, halogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(═O)—NH₂, —NO₂, —NH₂, and —OH; and n is aninteger from 0 to 4; with the proviso that R₃ is not chlorine orfluorine when R₁, R₄, R₅, R₁₁, and R₁₂ are hydrogen and R₁₀ and R₁₃ arechlorine.

In an embodiment of the fifth aspect, R₁ is hydrogen.

In an embodiment of the fifth aspect, R₁ is selected from the groupconsisting of Leu, Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH,—CH₂—C(═O)—(CH₂)C(CH₃)₂,

In an embodiment of the fifth aspect, R₃, R₄, R₅, R₉, R₁₀, R₁₁, R₁₂,R₁₃, and

R₁₄ are independently selected from the group consisting of hydrogen,chlorine, and fluorine.

In a sixth aspect, the compound of the fifth aspect is provided incombination with at least one pharmaceutically acceptable carrier ordiluent.

In a seventh aspect, a compound is provided having a formula selectedfrom the group consisting of:

wherein R₁ is selected from the group consisting of hydrogen, one aminoacid, two amino acids linked together, three amino acids linkedtogether,

R₆ is C₁₋₆ dialkyl amine; R₇ is selected from the group consisting ofhydrogen and C₁₋₆ alkyl; R₈ is C₁₋₆ alkyl; R₃, R₄, R₅, R₉, R₁₀, R₁₁,R₁₂, and R₁₃ are independently selected from the group consisting ofhydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(═O)—NH₂, —NO₂, —NH₂, and—OH; and n is an integer from 0 to 4.

In an embodiment of the seventh aspect, R₁ is hydrogen.

In an embodiment of the seventh aspect, R₁ is selected from the groupconsisting of Leu, Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH,—CH₂—C(═O) (CH₂)C(CH₃)₂,

In an embodiment of the seventh aspect, R₃, R₄, R₅, R₉, R₁₀, R₁₁, R₁₂,R₁₃, and R₁₄ are independently selected from the group consisting ofhydrogen, chlorine, and fluorine.

In an eighth aspect, the compound of the seventh aspect is provided incombination with at least one pharmaceutically acceptable carrier ordiluent.

In a ninth aspect, a compound is provided having a formula:

wherein R₁ is selected from the group consisting of hydrogen, one aminoacid, two amino acids linked together, three amino acids linkedtogether,

R₆ is C₁₋₆ dialkyl amine; R₇ is selected from the group consisting ofhydrogen and C₁₋₆ alkyl; R₈ is C₁₋₆ alkyl; R₃, R₄, R₅, R₉, R₁₀, R₁₁,R₁₂, R₁₃, and R₁₄ are independently selected from the group consistingof hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(═O)—NH₂, —NO₂, —NH₂,and —OH; and n is an integer from 0 to 4.

In an embodiment of the ninth aspect, R₁ is selected from the groupconsisting of Leu, Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH,—CH₂—C(═O)—(CH₂)C(CH₃)₂,

In an embodiment of the ninth aspect, R₃, R₄, R₅, R₉, R₁₀, R₁₁, R₁₂,R₁₃, and R₁₄ are independently selected from the group consisting ofhydrogen, chlorine, and fluorine.

In a tenth aspect, the compound of the ninth aspect is provided incombination with at least one pharmaceutically acceptable carrier ordiluent.

In an eleventh aspect, a compound is provided having a formula selectedfrom the group consisting of:

wherein R₁ is selected from the group consisting of hydrogen, one aminoacid, two amino acids linked together, three amino acids linkedtogether,

R₆ is C₁₋₆ dialkyl amine; R₇ is selected from the group consisting ofhydrogen and C₁₋₆ alkyl; R₈ is C₁₋₆ alkyl; R₃, R₄, R₅, R₉, R₁₀, R₁₁,R₁₂, and R₁₃ are independently selected from the group consisting ofhydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(═O)—NH₂, —NO₂, —NH₂, and—OH; and n is an integer from 0 to 4.

In an embodiment of the eleventh aspect, R₁ is hydrogen.

In an embodiment of the eleventh aspect, R₁ is selected from the groupconsisting of Leu, Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH,—CH₂—C(═O)—(CH₂)C(CH₃)₂,

In an embodiment of the eleventh aspect, R₃, R₄, R₅, R₉, R₁₀, R₁₁, R₁₂,R₁₃, and R₁₄ are independently selected from the group consisting ofhydrogen, chlorine, and fluorine.

In a twelfth aspect, the compound of the eleventh aspect is provided incombination with at least one pharmaceutically acceptable carrier ordiluent.

In a thirteenth aspect, a method for treating cancer in a mammal isprovided, comprising administering to the mammal an effective amount ofa compound selected from the group consisting of:

wherein R₁ is selected from the group consisting of hydrogen, one aminoacid, two amino acids linked together, three amino acids linkedtogether,

R₆ is C₁₋₆ dialkyl amine; R₇ is selected from the group consisting ofhydrogen and C₁₋₆ alkyl; R₈ is C₁₋₆ alkyl; R₃, R₄, R₅, R₉, R₁₀, R₁₁,R₁₂, R₁₃, and R₁₄ are independently selected from the group consistingof hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(═O)—NH₂, —NO₂, —NH₂,and —OH; and n is an integer from 0 to 4.

In an embodiment of the thirteenth aspect, R₃, R₄, R₅, R₉, R₁₀, R₁₁,R₁₂, R₁₃, and R₁₄ are independently selected from the group consistingof hydrogen, chlorine, and fluorine.

In an embodiment of the thirteenth aspect, a compound is provided withthe formula:

In an embodiment of the thirteenth aspect, the cancer comprises atranslocation gene fusion.

In an embodiment of the thirteenth aspect, the cancer is selected fromthe group consisting of Ewing's sarcoma, clear-cell sarcoma, myxoidliposarcoma, desmoplastic small round-cell tumor, myxoid chondrosarcoma,acute myeloid leukemia, congenital fibrosarcoma, prostate cancer andpancreatic cancer.

In an embodiment of the thirteenth aspect, the cancer is Ewing'ssarcoma.

In a fourteenth aspect, the compound of the thirteenth aspect isadministered in combination with another pharmaceutically active agent,either simultaneously, or in sequence.

In a fifteenth aspect, a use of a compound of the thirteenth aspect isprovided for the manufacture of a medicament for the treatment ofcancer.

Methods are also provided for screening agents that disruptprotein-protein interactions. Such methods include performingfluorescence polarization, and surface Plasmon resonance to detect suchagents. In some embodiments, the protein-protein interaction comprisesEWS-FLI1. In some embodiments, the EWS-FLI1 comprises a recombinantprotein. In other embodiments of methods for screening, theprotein-protein interaction can comprise a peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B provides data demonstrating the binding of recombinant RHA torecombinant EWS-FLI1 in ELISA. In FIG. 1A, standard 96 well ELISA plateswere coated with 500 ng EWS-FLI1 or BSA. Wells were incubated with 250ng RHA and bound RHA was detected with a polyclonal anti-RHA antibody.RHA bound specifically to EWS-FLI1 coated wells. When the experiment wasdone in the reciprocal order, RHA immobilized to surface, EWS-FLI1 boundspecifically to RHA. In FIG. 1B, wells were coated with 500 ng EWS-FLI1.Increasing concentrations of RHA (0.0133 nM to 13.3 nM) were incubatedin wells and detected by anti-RHA antibody.

FIGS. 2A-C provide data regarding identification of the region of RHAthat binds to EWS-FLI1. In FIG. 2A, recombinant EWS-FLI1 specificallybinds to fragment RHA (630-1020) compared to albumin (BSA) using anELISA assay. The RHA (630-1020) fragment is the one that contains thehomology to the peptide found to bind EWS-FLI1 by phage display libraryscreening. In FIG. 2B, a diagram is provided showing the binding sitesof proteins that bind to RHA. In FIG. 2C, GST-RHA fragments were used tocoimmunoprecipitate EWS-FLI1 from ESFT lysate (TC32 cells). GST proteinswere equalized using Coomassie staining. Glutathione conjugated beadswere mixed with GST-RHA fragments and TC32 cell lysate. Following PAGEresolution, EWS-FLI1 was detected with anti-FLI1 antibody.

FIG. 3A shows wild-type RHA augments EWS-FLI1 induced colony formation,while RHA mutant defective for EWS-FLI1 binding does NOT enhance colonygrowth. Murine embryonic fibroblasts were transfected with EWS-FLI1and/or RHA. EWS-FLI1 was expressed from the pBABE retroviral vector. RHAor mutants were expressed from pCMV-FLAG. The bars under the coloniesare the counts of triplicate soft-agar assays. Statistical analysis oftriplicate assays: EF+RHA versus EF+D827A p<0.001, EF+e.v. versusEF+either mutant, n.s. This experiment has been performed three timeswith independent transfections of plasmids. FIG. 3B shows that the D827Amutation did not affect RHA function. Blue bar represents a known RHAmutant, K417R. Immunoglobulin control immunoprecipitates did notdemonstrate ATPase activity. Phosphate standards were used to calibratethe assay and determine the rate of ATP hydrolysis. RHA (K417R) is aknown NTPase-null mutant of RHA.

FIG. 4 shows surface plasmon binding of the E9RP peptide to EWS-FLI1,where E9RP is the peptide region of RHA (E9R; SEQ ID NO: 29) fused tothe Penetratin peptide (a 16 aa long peptide from DrosophilaAntennapedia homeodomain protein (Antp) (SEQ ID NO: 34)) to achieveintracellular delivery of our peptides. Antp is conjugated to the aminoterminal of all peptides used in these studies. Recombinant EWS-FLI1 (MW55 kDa) was covalently linked to a CM5 chip at a density of 1200 RU, andE9RP (MW 4 kDa) was evaluated for binding. The E9RP analyteconcentrations ranged from 0.03-30 μM, and all are shown in duplicate.E9RP bound to EWS-FLI1 with a KD of 4.0 μM.

FIGS. 5A-B demonstrate that E9RP competes for RHA binding to EWS-FLI1 inELISA. E9RP is the peptide region of RHA (E9R; SEQ ID NO: 29) fused tothe Penetratin peptide (from the Antennapedia homeodomain; SEQ ID NO:34). In FIG. 5A, E9RP was used in a competition experiment for blockingbinding of RHA to EWS-FLI1. Wells were coated with 500 ng EWS-FLI1. RHA(4 nM) was added on to wells in the presence of E9RP (0.01-30 μM). InFIG. 5B, wells were coated with 500 ng/well recombinant CBP. RHA (4 nM)was added to wells with E9RP (0.01-30 μM).

FIG. 6 shows that E9RP disrupts RHA from binding to EWS-FLI1. E9RP isthe peptide region of RHA (E9R; SEQ ID NO: 29) fused to the Penetratinpeptide (from the Antennapedia homeodomain; SEQ ID NO: 34).Immunoprecipitation of GST-RHA using recombinant EWS-FLI1 bound to aFLI1 antibody. Lane 3 demonstrates the full complex while lanes 4through 7 show a dissociation of the complex using a 10 amino-acidpeptide, E9RP. E9RP is the sequence of aa 823-832 of human RHA that weresynthesized. Previous data show that this peptide binds to EWS-FLI1.

FIGS. 7A-B show that amino acids in positions 824 and 827 are importantfor EWS-FLI1 binding. FIG. 7A shows amino-acid positions 1, 2, 3, 5, and9 mutated to alanine in the GST-RHA (630-1020) and used in solutionimmunoprecipitation experiments similar to FIGS. 2 and 6. FIG. 7B showsthe results of densitometry of three immunoprecipitation experiments.Wild-type and mutations at position 1, 3, and 9 showed the expectedcomplex formation while mutations at positions 2 (P824A) and 5 (D827A)showed reduced complex formation. The graph shows a summary of threeexperiments using densitometry to quantify the amount of complexformation. *Student two-tailed t-test comparison of each mutant towild-type P824A p=0.0129 and D827A p=0.0344, others were notsignificant.

FIGS. 8A-E show E9RP inhibits ESFT cell growth. In FIGS. 8A and 8B, E9RPis the peptide region of RHA (SEQ ID NO:29) fused to the Penetratinpeptide (from the Antennapedia homeodomain; SEQ ID NO:34) and containsamino-terminal fluorescein (FITC). ESFT cell lines TC32, (ESFT 1), and5838 (ESFT 2) and a neuroblastoma cell line (SKNAS, NB, that lacksEWS-FLI1) demonstrated uptake of peptide at 24 hours following additionof peptide to culture media. Neither lipids nor electricity were usedfor transduction of peptides into cells. Confocal microscopy identifiedpeptide (green) throughout cells with nuclear uptake. Nuclear uptake wasconfirmed by DAPI staining. In FIG. 8C, two ESFT cell lines, 1 and 2,demonstrate reduced growth after seven days in culture, while the growthof the NB cells (lacking EWS-FLI1) is not affected (Row C). Cell lineswere placed in 96-well plates with either vehicle alone (, blue), orE9RP (10 μM) on days 0, 3 and 5 (▴, green). No lipid or othertransfection method was utilized. This represents two experiments withsimilar results. FIG. 8D depicts dose response of ESFT 1 to eitherwild-type or single amino-acid mutant peptides. (▪) wild-type E9RP, (E9R(SEQ ID NO:29) fused to the Penetratin peptide (SEQ ID NO:34)), (▾)(E9RP(P2A peptide (SEQ ID NO:30) fused to the Penetratin peptide (SEQ IDNO:34)), (▴)E9RP (D5A peptide (SEQ ID NO:31) fused to the Penetratinpeptide (SEQ ID NO:34)). Growth normalized to treating cells with the 16aa Antp sequence alone (SEQ ID NO:34). FIG. 8E is a comparison ofwild-type versus mutant peptides in ESFT and NB cells.

FIG. 9 shows expressed E9R reduced ESFT soft-agar growth, but notrhabdomyosarcoma growth. Plasmids that express E9R fused to enhancedgreen fluorescent protein (EGFP) were transfected into either Ewing'sSarcoma (TC71) or rhabdomyosarcoma (RP) cells and placed in soft-agarfor anchorage-independent growth. The pGE9R plasmid diffusely expressedE9R while the pGCE9R plasmid contained a nuclear export sequence toprevent expressed peptide from entering the nucleus. The transfectedcells were placed in soft agar culture for 2 weeks and stained with MTT.When E9R was diffusely expressed, including the nucleus, ESFT(containing EWS-FLI1) soft-agar colony growth was suppressed. ExpressedE9R did not affect the growth of rhabdomyosarcoma cells, which lackEWS-FLI1. Nuclear exclusion of expressed peptide did not decrease thegrowth of either cell line.

FIGS. 10A-C show expression of E9R (SEQ ID NO:29) peptide reduces ESFTxenografts growth. ESFT cells TC71 were transfected with pGE9R(wild-type sequence peptide (SEQ ID NO: 29)) or pGE9R-D5A (control,mutated peptide; D5A (SEQ ID NO: 31)). In FIG. 10A, fluorescentmicrographs of transfected cells after 4 days in culture are presented.The first panel shows brightfield cells, the second with green filter,and the third, magnification of a representative area. Bar equals 300microns. In FIG. 10B, each group contains six mice. Mice legs weremeasured every 2-3 days. Tumors appeared in 2 weeks. The experiment wasterminated after 26 days when the largest tumor caused the mouse tolimp. Tumor volumes were calculated according to (D×d2/6)×π formula,where large diameter of tumor mass is D and small diameter is d. 2-wayANOVA analysis, followed by Bonferroni correction for multiplevariables, the difference in the two growth curves was highlysignificant, p=0.016. FIG. 10C is a demonstration of orthotopic site ofrepresentative mice after 26 days.

FIG. 11 relates to small molecule libraries that contain EWS-FLI1binding molecules and shows the results of two SPR screens using BiacoreT100. This demonstrates the type of results obtained from screening 80compounds from the NCI DTP. The values are the ratio of the resonanceunits obtained divided by the expected resonance maximum. Theconsistency of two independent experiments is shown by the overlap inred and blue. Each number on the x-axis represents one tested compound.

FIG. 12 shows surface plasmon binding of NSC635437 to EWS-FLI1 with a KDof 2.34 μM.

The red lines are the actual binding curves while the thin black linesare the curve fit overlays.

FIG. 13 shows that NSC635437 decreases RHA binding to EWS-FLI1 anddemonstrates specific toxicity. In FIG. 13A, recombinant EWS-FLI1 wastreated with NSC635437 for 30 minutes. GST-RHA (630-1020) was added andthe complexes were then precipitated with antibody to EWS-FLI1 andProtein G beads. Resolved complexes showing the immunoprecipitatedcomplex control without compound are in lanes 3 (0.1% DMSO) and 5 (0.01%DMSO). Addition of NSC635437, shown in lanes 4 (10 μM) and 6 (1 μM),decreased complex formation compared to DMSO matched controls in lanes 3and 5, respectively. FIG. 13B shows NSC635437 reduces ESFT but notneuroblastoma cell growth. ESFT TC32 (◯) and neuroblastoma SK-N-AS (▪)were grown for 72 hours in the continuous presence of NSC635437. Cellswere incubated in MTT reagent for 4 hours and the dried precipitate wasdissolved isopropanol and the absorbance read at 570 nm. Each cell linewas standardized to the growth of the DMSO control.

FIG. 14A shows the BLAST Alignment for peptide motif E9R PPPLDAVIEA(E9R; SEQ ID NO: 29). The best sequence alignment was extracted and thestructure was predicted using homology modeling with aSQUALENE-HOPENE-CYCLASE (PDB: 1SQC) x-ray structure as a template. Theinput alignment for the Modeler was obtained with BLAST. FIG. 14B showsthe BLAST Alignment for peptide sequence motif ‘PPPLD’ (SEQ ID NO: 32)with root mean square deviation (RMSD, black) of their structure incomparison to query. In the ligand overlap strategy, the Brookhavendatabase for x-ray structures that contained the PPPLDAVIEA (E9R; SEQ IDNO: 29) motif was sampled, a BLAST alignment was made for the 10-merPPPLDAVIEA sequence (E9R; SEQ ID NO: 29) to predict the structure.

FIG. 15 shows modeling of E9R with existing proteins and NSC635437. FIG.15A shows the PPPLDAVIEA (E9R; SEQ ID NO: 29) motif (in green)superimposed with the structures of ‘PPPLD’(SEQ ID NO: 32) sequencefinger print retrieved from the Protein database. FIG. 15B showsPPPLDAVIEA (E9R; SEQ ID NO: 29) peptide RMS fit with NSC635437. Ligandcarbon atoms are colored green. FIG. 15C shows a pharmacophore model forlibrary screening using the PPPLD motif.

FIG. 16 shows synthesis of3-hydroxy-3-(2-oxo-2-phenyl-ethyl)-1,3-dihydro-indol-2-one (30).

FIG. 17 shows schema for EWS-FLI1 small molecule and peptidedevelopment.

FIG. 18 shows planned modifications of compound NSC635437.

FIG. 19 shows retrosynthesis of the key isatin core.

FIG. 20A shows a model synthesis for enantiomers R-(−)-31 and S-(+)-31,and an x-ray structure of R-(−)-31. FIG. 20B shows NMR spectroscopy ofthe R-(−)-31 enantiomer.

FIG. 21 shows the addition of the Reformasky reagent in the presence of1.5 equivalents of cinchonine and 4.0 equivalents of pyridine toaromatic ketone to yield a tertiary alcohol in 97% yield and 97% ee.

FIG. 22 shows the synthesis of non-racemic isatin derivatives byenantioselective Reformatsky-like reaction.

FIG. 23 shows results of surface plasmon resonance for various compoundsbinding to EWS-FLI1. Each trace represents a different concentration ofthe tested compound. For the control NSC635437 compound, a KD of2.10E-06 M was measured. For the YK-4-275 compound, a KD of 2.34E-07 Mwas measured. For the YK-4-280 compound, a KD of 1.35E-06 M wasmeasured. For the YK-4-284 compound, a KD 1.30E-06 M was measured. Forthe YK-4-289 compound, a KD of 6.82E-06 M was measured.

FIG. 24 shows results of surface plasmon resonance for various compoundsbinding to EWS-FLI1. Each trace represents a different concentration ofthe tested compound. For the control NSC635437 compound, a KD of1.21E-06 M was measured. For the YK-4-279 compound, a KD of 4.56E-06 Mwas measured. For the YK-4-276 compound, a KD of 1.02E-05 M wasmeasured. For the YK-4-284 compound, a KD of 1.28E-05 M was measured.For the YK-4-289 compound, a KD of 9.30E-06 M was measured. For thecompound YK-4-275, a KD of 2.02E-06 M was measured. For the YK-4-280compound a KD of 6.39E-06 M was measured.

FIG. 25 shows the summary of three EF-RHA IP experiments. FIG. 25demonstrates the effect of each of the YK-4-XXX and NSC635437 compounds(where XXX is the compound number labeled on the x-axis) upon RHAbinding to EWS-FLI1. Each of the YK-4-XXX compounds was added torecombinant EWS-FLI1 at a final concentration of 10 microM. GST-RHA(630-1020) was added and the complex was immunoprecipitated with rabbitantiserum against FLI1. Complexes were resolved used PAGE and the amountof GST-RHA (630-1020) that co-precipitated was quantified usingdensitometry. Three experiments were averaged to obtain the datapresented in the figure. The lower the value, the more effective thesmall molecule at disrupting EWS-FLI1 binding to RHA.

FIG. 26 shows IC50 on Ewing's sarcoma cells. This figure demonstratesthe effects of each of the YK-4-XXX compounds (where XXX is the compoundnumber labeled on the x-axis) upon the growth of ESFT cell line TC-32.Cells were added to triplicate wells of a 96-well plate. Twenty-fourhours after adding cells, compounds were added to the wells in at thedoses indicated. Seven days after plating the cells, the dye WST wasadded to cells and following a 3-hour incubation, the absorbanceindicated the relative cell number.

FIG. 27 shows toxicity of peptides on the TC32 (ESFT) and HEK (control)cell lines. Both cell lines were treated at 0, 3 and 5 days, with 30 μMpeptides corresponding to SEQ ID NO.s: 1-27 (See Table 1). Cellviability was measured by MTT on day 7. Water was used as a control.

FIG. 28 shows inhibition of tumor growth in mice. Nude mice wereinoculated with ESFT cell line CHP-100. When tumors were palpable,animals were treated with YK-4-275 (NSC635437) for 4 days. Curves showtumor volumes over time.

FIG. 29A shows a schematic representation of RHA including the regionthat binds to EWS-FLI1. The E9R peptide corresponds to amino acids 823to 832, located in the proximal of HA2 region of RHA. FIG. 29B shows aNorthern blot for an shRNA expression vector transfected into TC71(ESFT) cells to reduce RHA levels. FIG. 29C shows TC71 viability wasreduced, as measured by WST reduction, following RHA shRNA expression.FIG. 29D shows a graph of alanine mutagenesis within E9R sequencefollowed by in vitro immunoprecipitation with EWS-FLI1. The density ofthe GST-RHA band was measured and this graph is the average of threeexperiments. RHA P824A and D827A mutants have significantly lowerbinding to EWSFLI1 (*p=0.0129 and **p=0.0034 respectively). FIG. 29Eshows the results of murine fibroblasts placed in soft-agar foranchorage-independent growth assays (empty vector (W), EWS-FLI1 alone(WEF1)). FIG. 29F shows a graph that enumerates the colonies counted inthree separate experiments; the difference between wild-type and mutantRHA was significant (*p=0.0028). FIG. 29G shows protein expression forthe fibroblasts, detected with anti-FLAG (top) or anti-FLI1 (bottom).FIG. 29H shows a graph of densitometry of the EWSFLI1 blot performedusing MultiGauge software.

FIG. 30 provides data to show E9R peptide prevents EWS-FLI1 binding toRHA with specific detrimental effects upon ESFT growth andtransformation. FIG. 30A shows a Western blot for immunoprecipitation ofGST-RHA (647-1075) using recombinant full-length EWS-FLI1 bound to aFLI1 antibody. FIG. 30B shows a graph for growth reduction upon E9R-P(Antennapedia-E9R) treatment (10 μM) was observed in TC32 cells but notSKNAS cells. FIG. 30C shows photomicrographs for E9R-P peptide uptaketracked with FITC label (upper panels). Merged images of DAPI nuclearcounter-stain (middle panels) and FITC-Ant-E9R (lower panels) showedpeptide was distributed throughout the cytoplasm and nucleus of bothTC32 and SKNAS cells. Scale bar equals 20 μm. FIG. 30D shows a graphwhere neither Antennapedia alone (Antp), nor a mutant of an asparticacid residue important for RHA binding to EWS-FLI1 (E9R-D5A-P) reducedgrowth of TC32 cells while E9R-P reduced cell growth. FIG. 30E showsphotomicrographs for TC71 and SKNAS cells expressed EGFP empty vector(pG), EGFP-E9R (pGE9R), EGFP with nuclear export sequence (pGC) orEGFP-E9R with nuclear export sequence (pGCE9R). Only expression ofEGFP-E9R in TC71 reduced anchorage independent growth. FIG. 30F shows agraph for colony numbers of three experiments averaged with asignificant reduction in only TC71 cells when expressing E9R throughoutthe cell (*p=0.0012). Scale bar equals 20 μm.

FIG. 31 provides data showing a small molecule binding to EWS-FLI1 anddisplaces E9R from EWS-FLI1. FIG. 31A shows NSC635437,3-hydroxy-3-(2-oxo-2-phenyl-ethyl)-1,3-dihydro-indol-2-one synthesizedwith 100% yield. Aromatic functionalization produced YK-4-279 apara-methoxy derivative of NSC635437. FIG. 31B shows a Western blot forEWS-FLI1 incubated with NSC635437 (left panel) or YK-4-279 (right panel)followed by the addition of GST-RHA (647-1075). A FLI1 antibodycomplexed EWS-FLI1 and precipitated it from the solution. FIG. 31C showsa graph for YK-4-279 steady state kinetics for binding to recombinantEWS-FLI1 that was immobilized on a CM5 Biacore chip. FIG. 31D shows agraph for SPR displacement assay of 64 μM E9R alone (Black solid line)and with addition of YK-4-279 (Grey dashed line); 32 μM E9R alone (Darkblue solid line) and with addition of YK-4-279 (Light blue dashed line).FIG. 31E shows a graph for fluorescent polarization indicated thebinding of 3.2 μM of FITC-E9R to EWS-FLI1, which was competitivelyinhibited by increasing concentrations of YK-4-279.

FIG. 32 provides data showing YK-4-279 reduces EWS-FLI1 functionalactivity. FIG. 32A concerns TC32 cells that were treated with YK-4-279and resolved protein lysates and that were immunoblotted forco-precipitated RHA (top), EWS-FLI1 (middle), or total RHA (bottom).FIG. 32B shows a graph for a luciferase reporter assay of EWS-FLI1responsive NROB1 promoter showed YK-4-279 dose-dependent (18-hourtreatment) reduction in the promoter activity in COS7 cells. FIG. 32Cshows a Northern blot for protein lysates from transfected cells showedexpression of EWS-FLI1. FIG. 32D shows a Northern blot for YK-4-279treated TC32 cell lysates (treated for 14 hours) were blotted for cyclinD1 and actin.

FIG. 33 provides data showing YK-4-279 is potent and specific inhibitorof ESFT. FIG. 33A shows a graph for TC32 cells treated with a dose rangeof YK-4-279 and NSC635437. Cell number was measured by MTT or WSTreduction after seven days in culture. FIG. 33B shows a graph for TC32and HEK-293 (non-transformed, lacking EWS-FLI1) treated similarly to(A). FIG. 33C shows a graph for primary ESFT explant cell lines GUES1and ES925 treated for 3 days with YK-4-279. FIG. 33D cell linesexpressing EWS-FLI1 compared to non-EWS-FLI1 malignant cell linesfollowing 3 days in culture to establish the IC50 using WST assay. FIG.33E shows a graph for caspase 3 activity of a panel of ESFT (TC32, TC71,A4573, and ES925), malignant non-EWS-FLI1 expressing (MCF-7, MDA-MB-231,PC3, ASPC1, COLO-PL), and non-transformed cells (HEK-293, HFK, and HEC).Graph plots level of fluorescence in treated divided by untreatedlysate. FIG. 33F shows photomicrographs where arrows indicated apoptoticnuclear fragmentation after 50 μM YK-4-279 treated ESFT (TC32) andnon-transformed cells (HEK-293, HFK, and HEC). Scale bar equals 200 μm.

FIG. 34 provides data showing YK-4-279 inhibited the growth of ESFTxenograft tumors. Xenografts were established with injection of eitherESFT (CHP-100 or TC71) or Prostate cancer (PC3) cells. FIG. 34A shows agraph for CHP-100 intramuscular xenografts (arrow indicates when tumorswere palpable) receiving DMSO (n=4; open circles) or 1.5 mg YK-4-279(n=5; triangles) (p=0.016, by t Test comparison). Single experimentgrowth curves depicted are representative of five independentexperiments. FIG. 34B shows a graph for PC3 subcutaneous xenografts(arrow indicates when tumors were palpable) treated as CHP-100 cells (a)(n=5 per group, representative of 3 independent experiments). FIG. 34Cshows a graph for overall response of ESFT xenografts (TC71, opensymbols, and CHP-100, closed symbols) to YK-4-279 (1.5 mg/dose). Tumorvolumes at day 14 after treatment initiation compared across 5experiments (DMSO n=19, YK-4-279 n=25, p<0.0001, by Mann-Whitney test).FIG. 34D shows a photomicrograph for tumors from the mice in (A)analyzed for activation of caspase-3 activity usingimmunohistochemistry. FIG. 34E shows a graph for caspase-3 positivecells counted (n>500 in 3 high-power-fields) in 4 separately stainedslides for each group (p=0.041).

FIG. 35 provides data showing PANC1 cells infected with virus containingeither siRNA for RHA or control luciferase. FIG. 35A shows an immunoblotshowing protein levels following 6 days of selection. FIG. 35B showsviability of cells using WST reduction 6 days after selection and RHAreduction.

FIG. 36 shows a graph for ATPase assay where Biomol Green was used todetect free phosphate. While the P824A mutant did show reduced ATPaseactivity, the D827A mutation did not affect RHA function. Phosphatestandards were used to calibrate the assay and determine the rate of ATPhydrolysis. RHA (K417R) is a known NTPase-null mutant of RHA.Immunoglobulin control immunoprecipitations did not demonstrate ATPaseactivity.

FIG. 37 shows an immunoblot from log-phase cell lysates that were eithertreated with DMSO control or 10 μM YK-4-279 overnight.

FIG. 38A provides data showing COST cells transfected with an NFκBreporter construct followed by stimulation with PMA. Cells were treatedwith YK-4-279 following PMA treatment. Cell lysates were analyzed forNFκB induced luciferase activity as standardized to LTR activatedrenilla luciferase. FIG. 38B shows an immunoblot from log-phase celllysates that were either treated with DMSO control or 10 μM YK-4-279overnight. FIG. 38C shows a graph of densitometry of treated/untreatedwith both standardized for β-tubulin expression.

FIG. 39A provides data showing HEC and HFK, non-transformed endocervicalcells and keritinocytes, treated with YK-4-279 for 72 hours and assayedfor viability using WST reduction. FIG. 39B provides data showing TC71cells treated for 16 hours with YK-4-279 or doxorubicin. Lysates wereassayed for cleavage of AMC-DEVD by induced caspase-3 and fluorescencewas measured. FIG. 39C provides data showing that RHA reduced TC71 cellswere more resistant to YK-4-279 treatment than wild-type cells. FIG. 39Dprovides data showing an shRNA tet-inducible expression vector stablytransfected into A673 (ESFT) cells to reduce EWS-FLI1 levels. FIG. 39Eprovides data showing that EWS-FLI1 reduced A673 cells were moreresistant to YK-4-279 treatment than wild-type cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description and examples illustrate some exemplaryembodiments of the disclosed invention in detail. Those of skill in theart will recognize that there are numerous variations and modificationsof this invention that are encompassed by its scope. Accordingly, thedescription of a certain exemplary embodiment should not be deemed tolimit the scope of the present invention.

Overview

The greater the tumor target specificity the more likely that successfulagents will be effective and lack non-specific toxicity. Many tumors,particularly chemotherapy-resistant leukemias and sarcomas, containtumor-specific chromosomal translocations that encode fusion proteinsthat are present only in the tumor. The Ewing's Sarcoma Family of Tumors(ESFT) contains a characteristic translocation, t(11:22), which leads tothe oncogenic transcription factor EWS-FLI1 (synonyms include:EWS/FLI-1; EWS-FLI-1). EWS-FLI1 is a critical tumor-specific oncogene inpatients with ESFT because it is derived from a chromosomaltranslocation and is necessary to maintain tumor growth.

RNA helicase A (RHA), a member of the DEXH box helicase family ofproteins, is an integral component of protein complexes that regulatetranscription, splicing and mRNA translation in a distinct class ofproteins. A novel direct protein-protein interaction between RHA andEWS-FLI1 by phage display library screening has been demonstrated.EWS-FLI1 oncoprotein is expressed as a result of a chromosomaltranslocation that occurs in patients with Ewing's Sarcoma Family ofTumors (ESFT). Although more than 95% of the tumors carry EWS-FLI1,therapeutic applications using this target have not been developed.EWS-FLI1 and RHA interact to promote and maintain the oncogenicphenotype of ESFT. Endogenous and direct interaction of RHA and EWS-FLI1both in ESFT cell lines and with recombinant proteins has been observed.Chromatin immunoprecipitation experiments demonstrated both proteinsbound to EWS-FLI1 target gene promoters. RHA stimulated thetranscriptional activity of EWS-FLI1 regulated promoters, including Id2,in ESFT cells. In vitro mutagenesis of each aa 823-832 of RHA intoalanine prevented EWS-FLI1 binding only with mutations of amino acidsP824A and D827 A. Wild-type RHA expression in MEF cells stablytransfected with EWS-FLI1 enhanced the anchorage-independent phenotypecompared to EWS-FLI1 alone. When MEF cells are transfected with RHA(K417R), abolishing ATPase activity, diminished anchorage-independentgrowth is observed. When RHA (P824A) or RHA (D827A) are transfected intoEWS-FLI1 expressing MEFs, there was no increase in anchorage independentgrowth over empty vector transfected cells. The E9R peptide was alsoevaluated for the ability to disrupt EWS-FLI1 from RHA. The peptidecaused dissociation of EWS-FLI1 from GST-RHA (630-1020). The E9R of RHAwas cloned into a vector expressing the peptide as a fusion protein withEGFP. The peptide-EGFP chimera significantly reducedanchorage-independent growth when present in the nucleus, but not whenpresent exclusively in the cytoplasm. When the peptide was expressed inrhabdomyosarcoma cells, colony formation was not affected. Reduction ofRHA protein levels by siRNA in ESFT cell lines also decreased theirgrowth rate. These results indicate that the EWS-FLI1 interaction withRHA is important for the oncogenic function of EWS-FLI1.

Since EWS-FLI1 lacks intrinsic enzymatic activity, one approach topharmacologic inhibition is through inhibition of protein-proteininteraction. RNA Helicase A (RHA, p150), a DEAD/H family member thatmodulates gene expression, has been identified as a critical partner ofEWS-FLI1. EWS-FLI1 binds to a unique region of RHA that is not involvedin non-malignant RHA transcriptional modulation. A compound has beenidentified that blocks EWS-FLI1 interaction with RHA. The interactionthat is targeted is that of RHA with EWS-FLI1, which results in a potenttranscriptional activator/coactivator complex, which amplifies thefunctions of both proteins and drives the malignant phenotype of ESFT.

EWS-FLI1 is a very promising ESFT molecular target since it is relevantin patients, required for growth, and specific to tumors. Therapiesdirected towards the inactivation of EWS-FLI1 can address thesignificant problem of recurrent disease for patients. Reagents havebeen developed that disrupt the interaction of EWS-FLI1 with RHA andthus provide new therapeutic agents. Also provided are additional smallmolecules that bind and inhibit EWS-FLI1, and thus have utility intherapeutic agents.

The molecular signature, cellular biology, and anti-tumor effect ofblocking EWS-FLI1 from binding to RNA Helicase A (RHA) has beendetermined, and peptides have been developed that bind to EWS-FLI1,block RHA from interacting with EWS-FLI1, and lead to ESFT-specific celltoxicity. Small molecules that exert a similar function have also beenidentified by screening the NCI DTP library of compounds for moleculesthat bind to EWS-FLI1. The small molecules have significant3-dimensional homology to the first 3 amino acids of the functionalpeptide, E9R.

The therapeutic agents of preferred embodiments have broad applicabilityto a larger group of tumors, and are useful as therapeutics for othertranslocation fusion-protein defined malignancies such aschemotherapy-resistant sarcomas and leukemias, and other difficult totreat tumors, including ESFT. These therapeutic agents offer enhancedspecificity of treatment leading to reduced mortality and morbidity.

Small Molecule EWS-FLI1-Protein Inhibitors

Small molecule EWS-FLI1 protein inhibitors include compounds of thefollowing structure:

In the above structure, R₁ is a substituent selected from the groupconsisting of hydrogen, from one to three amino acids (e.g. Leu,Leu-Asp, Leu-Asp-Ala),

wherein R6 is C₁₋₆ dialkyl amine; R7 is selected from the groupconsisting of hydrogen and C1-6 alkyl; R8 is C1-6 alkyl; R3, R4, R5, R9,R10, R11, R12, R13, and R14 are independently selected from the groupconsisting of hydrogen, halogen (e.g., F, Cl, Br, I), C1-6 alkyl (e.g.,—CH₃), C₁₋₆ alkoxy (e.g. —OCH₃), —C(═O)NH2, —NO2, —NH2, and —OH; and nis an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4); with the proviso thatR3 is not chlorine or fluorine when R1, R4, R5, R11, and R12 arehydrogen and R10 and R13 are chlorine.

Small molecule EWS-FLI1 protein inhibitors also include compounds of thefollowing structure:

In the above structure, R₁ is selected from the group consisting ofhydrogen, from one to three amino acids (e.g. Leu, Leu-Asp,Leu-Asp-Ala),

wherein R6 is C1-6 dialkyl amine; R7 is selected from the groupconsisting of hydrogen and C1-6 alkyl; R8 is C1-6 alkyl; R3, R4, R5, R9,R10, R11, R12, R13, and R14 are independently selected from the groupconsisting of hydrogen, halogen (e.g., F, Cl, Br, I), C1-6 alkyl (e.g.—CH3), C1-6 alkoxy (e.g., —CH₃), —C(═O)NH2, —NO2, —NH2, and —OH; and nis an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4).

One subset of preferred compounds includes compounds with the formula:

In the above structures, R₁ is selected from the group consisting ofhydrogen, from one to three amino acids (e.g. Leu, Leu-Asp,Leu-Asp-Ala),

wherein, R6 is C1-6 dialkyl amine; R7 is selected from the groupconsisting of hydrogen and C1-6 alkyl; R8 is C1-6 alkyl; R3, R4, R5, R9,R10, R11, R12, and R13 are independently selected from the groupconsisting of hydrogen, halogen (e.g., F, Cl, Br, I), C1-6 alkyl (e.g.,—CH3), C1-6 alkoxy (e.g. —OCH3), —C(═O)NH2, —NO2, —NH2, and —OH; and nis an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4).

Yet another subset of preferred compounds includes compounds of thefollowing formula:

In the above structures, R₁ is selected from the group consisting ofhydrogen, from one to three amino acids (e.g. Leu, Leu-Asp,Leu-Asp-Ala),

wherein R₆ is C₁₋₆ dialkyl amine; R₇ is selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; R₈ is C₁₋₆ alkyl; R₃, R₄, R₅, R₉,R₁₀, R₁₁, R₁₂, and R₁₃ are independently selected from the groupconsisting of hydrogen, halogen (e.g., F, Cl, Br, I), C₁₋₆ alkyl (e.g.,—CH₃), C₁₋₆ alkoxy (e.g. —OCH₃), —C(═O)NH₂, —NO₂, —NH₂, and —OH; and nis an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4).

A particularly preferred compound for use in treating cancers includingthe Ewing's sarcoma family of tumors, pancreatic cancer, prostatecancer, and other cancers comprising translocation gene fusions has theformula:

A preferred subset of small molecule EWS-FLI1 protein inhibitors includecompounds of the following structures:

In the above structures, X is carbon or nitrogen; R₁ is a substituentselected from the group consisting of hydrogen, from one to three aminoacids (e.g., Leu, Leu-Asp, Leu-Asp-Ala),

wherein R₆ is a C₁₋₆ dialkyl amine, R₇ is hydrogen or C₁₋₆ alkyl, and R₈is C₁₋₆ alkyl; R₃, R₄, R₅, R₁₀, R₁₁, R₁₂, and R₁₃ are independentlyselected from the group consisting of hydrogen, halogen (e.g., F, Cl,Br, I), C₁₋₆ alkyl (e.g., —CH₃), C₁₋₆ alkoxy (e.g., —OCH₃), —C(═O)NH₂,—NO₂, —NH₂, and —OH; and n is an integer from 0 to 4 (e.g., 0, 1, 2, 3,4); with the proviso that R₃ is not chlorine or fluorine when thecompound includes a hydroxy group on the carbon atom of the bicyclicring system that links the bicyclic ring system to the monocyclic ringvia the linking group, X is carbon, R₁, R₄, R₅, R₁₁, and R₁₂ arehydrogen and R₁₀ and R₁₃ are chlorine.

Another subset of preferred compounds includes those of the formulas:

In the above structures, X is carbon or nitrogen; R₁ is a substituentselected from the group consisting of hydrogen, from one to three aminoacids (e.g., Leu, Leu-Asp, Leu-Asp-Ala),

wherein R₆ is a C₁₋₆ dialkyl amine, R₇ is hydrogen or C₁₋₆ alkyl, and R₈is C₁₋₆ alkyl; R₃ is selected from the group consisting of hydrogen,bromine, iodine, C₁₋₆ alkyl (e.g., —CH₃), C₁₋₆ alkoxy (e.g., —OCH₃),—C(═O)NH₂, —NO₂, —NH₂, and —OH; R₄, R₅, R₁₀, R₁₁, R₁₂, and R₁₃ areindependently selected from the group consisting of hydrogen, halogen(e.g., F, Cl, Br, I), C₁₋₆ alkyl (e.g., —CH₃), C₁₋₆ alkoxy (e.g.,—OCH₃), —C(═O)NH₂, —NO₂, —NH₂, and —OH; and n is an integer from 0 to 4(e.g., 0, 1, 2, 3, 4).

Another subset of preferred compounds includes those of the formulas:

In the above structures, X is carbon or nitrogen; R₁ is a substituentselected from the group consisting of hydrogen, from one to three aminoacids (e.g., Leu, Leu-Asp, Leu-Asp-Ala),

wherein R₆ is a C₁₋₆ dialkyl amine, R₇ is hydrogen or C₁₋₆ alkyl, and R₈is C₁₋₆ alkyl; R₃, R₄, R₅, R₁₁, and R₁₂ are independently selected fromthe group consisting of hydrogen, halogen (e.g., F, Cl, Br, I), C₁₋₆alkyl (e.g., —CH₃), C₁₋₆ alkoxy (e.g., —OCH₃), —C(═O)NH₂, —NO₂, —NH₂,and —OH; R₁₀ and R₁₃ are independently selected from the groupconsisting of hydrogen, bromine, iodine, C₁₋₆ alkyl (e.g., —CH₃), C₁₋₆alkoxy (e.g., —OCH₃), —C(═O)NH₂, —NO₂, —NH₂, and —OH; and n is aninteger from 0 to 4 (e.g., 0, 1, 2, 3, 4).

Yet another subset of preferred compounds includes those of thefollowing formula:

wherein R₁ is a substituent selected from the group consisting ofhydrogen, from one to three amino acids (e.g., Leu, Leu-Asp,Leu-Asp-Ala),

wherein R₆ is a C₁₋₆ dialkyl amine, R₇ is hydrogen or C₁₋₆ alkyl, and R₈is C₁₋₆ alkyl; R₃, R₄, R₅, R₁₁, and R₁₂ are independently selected fromthe group consisting of hydrogen, halogen (e.g., F, Cl, Br, I), C₁₋₆alkyl (e.g., —CH₃), C₁₋₆ alkoxy (e.g., —OCH₃), —C(═O)NH₂, —NO₂, —NH₂,and —OH; R₁₀ and R₁₃ are independently selected from the groupconsisting of hydrogen, bromine, iodine, C₁₋₆ alkyl (e.g., —CH₃), C₁₋₆alkoxy (e.g., —OCH₃), —C(═O)NH₂, —NO₂, —NH₂, and —OH; and n is aninteger from 0 to 4 (e.g., 0, 1, 2, 3, 4).

Another subset of preferred compounds includes those of the followingformulas.

wherein R₁ is a substituent selected from the group consisting ofhydrogen, from one to three amino acids (e.g., Leu, Leu-Asp,Leu-Asp-Ala),

wherein R₆ is a C₁₋₆ dialkyl amine, R₇ is hydrogen or C₁₋₆ alkyl, and R₈is C₁₋₆ alkyl; R₁₁, and R₁₂ are independently selected from the groupconsisting of hydrogen, halogen (e.g., F, Cl, Br, I), C₁₋₆ alkyl (e.g.,—CH₃), C₁₋₆ alkoxy (e.g., —OCH₃), —C(═O)NH₂, —NO₂, —NH₂, and —OH; R₁₀and R₁₃ are independently selected from the group consisting ofhydrogen, bromine, iodine, C₁₋₆ alkyl (e.g., —CH₃), C₁₋₆ alkoxy (e.g.,—OCH₃), —C(═O)NH₂, —NO₂, —NH₂, and —OH; and n is an integer from 0 to 4(e.g., 0, 1, 2, 3, 4).

Another subset of preferred compounds includes those of the followingformulas.

wherein R₁ is a substituent selected from the group consisting ofhydrogen, from one to three amino acids (e.g., Leu, Leu-Asp,Leu-Asp-Ala),

wherein R₆ is a C₁₋₆ dialkyl amine, R₇ is hydrogen or C₁₋₆ alkyl, and R₈is C₁₋₆ alkyl; R₃, R₄, and R₅, are independently selected from the groupconsisting of hydrogen, halogen (e.g., F, Cl, Br, I), C₁₋₆ alkyl (e.g.,—CH₃), C₁₋₆ alkoxy (e.g., —OCH₃), —C(═O)NH₂, —NO₂, —NH₂, and —OH; and nis an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4).

Particularly preferred compounds also include those of the followingformulas.

wherein R₁ is a substituent selected from the group consisting ofhydrogen, from one to three amino acids (e.g., Leu, Leu-Asp,Leu-Asp-Ala),

wherein R₆ is a C₁₋₆ dialkyl amine, R₇ is hydrogen or C₁₋₆ alkyl, and R₈is C₁₋₆ alkyl; and n is an integer from 0 to 4 (e.g., 0, 1, 2, 3, 4).

In preferred embodiments of the above subsets, R₁ is hydrogen. Otherpreferred substituents for R₁ include —CH₂—C(═O)—NHCH₂COOH,CH₂—C(═O)—(CH₂)C(CH₃)₂, Leu-Asp, Leu-Asp-Ala, Leu,

Other preferred substituents include R₃ as halogen (e.g., F, Cl, Br, I),C₁₋₆ alkyl (e.g., —CH₃), C₁₋₆ alkoxy (e.g., —OCH₃), —NO₂, NH₂, or —OH,and R₄ and R₅ as hydrogen. Alternatively, other preferred substituentcombinations include R₄ as chloro and R₃ and R₅ as hydrogen; or R₅ aschloro and R₃ and R₄ as hydrogen; or R₄ and R₃ as chloro and R₅ ashydrogen; or R₃, R₄ and R₅ as chloro. It is particularly preferred tohave R₄ and R₅ as hydrogen, with a non-hydrogen substituent as R₃ (parasubstitution), although ortho and meta substitution are also acceptable.Particularly preferred compounds are disubstituted (two non-hydrogensubstituents amongst R₃, R₄ and R₅) and trisubstituted (R₃, R₄ and R₅are each non-hydrogen substituents).

Certain compounds of preferred embodiments include a chiral center atthe point of attachment of the hydroxy substituent. Both the R and the Senantiomers can be prepared. A method of preparing the enantiomericforms for the core ring structure is provided in FIG. 20, and can beadapted to prepare desired enantiomers preferentially.

Compounds of the above structures can be prepared according to thefollowing synthesis schemes.

In these schemes, ketone (4.0 equiv.) and a catalytic amount ofdiethylamine (10 drops) are added to a solution of substituted isatin(1.0 equiv.) in methanol (5 mL). The mixture is stirred at roomtemperature until starting material (substituted isatin) disappearscompletely. The resulting solution is concentrated and applied to flashchromatography eluting with hexane/ethyl acetate to afford pure productin quantitative yield. Further purification is done by recrystallizationwith hexane/ethyl acetate.

The inhibitors incorporating a carbon-carbon double bond in the grouplinking the two ring systems can be prepared from the correspondingsaturated inhibitor by reducing the compound using synthetic techniquesknown in the art.

Compounds of the following structures were prepared according to theabove-references synthesis scheme.

NMR spectra were recorded for the compounds using a Varian-400spectrometer for ¹H (400 MHz). Chemical shifts (δ) are given in ppmdownfield from tetramethylsilane as internal standard, and couplingconstants (J-values) are in hertz (Hz). Purifications by flashchromatography were performed. The results were as follows.

4,7-Dichloro-3-[2-(4-chlorophenyl-2-oxoethyl)]-3-hydroxyl-1,3-dihydroindol-2-one(YK-4-275): white solid; mp 194-196° C.; ¹H NMR (DMSO, 400 MHz) δ 10.96(s, 1H), 7.93 (d, 2H, J=8.8 Hz), 7.57 (d, 2H, J=8.8 Hz), 7.30 (d, 1H,J=8.8 Hz), 6.90 (d, 1H, J=8.8 Hz), 6.47 (s, 1H), 4.36 (d, 1H, J=18.4Hz), 3.71 (d, 1H, J=18.0 Hz).

1-Benzyl-4,7-dichloro-3-[2-(4-chlorophenyl-2-oxoethyl)]-3-hydroxy-1-1,3-dihydroindol-2-one(YK-4-276): white solid; mp 154-156° C.; ¹H NMR (DMSO, 400 MHz) δ 7.94(d, 2H, J=8.8 Hz), 7.56 (d, 2H, J=8.8 Hz), 7.30 (m, 4H), 7.25 (d, 2H,J=8.8 Hz), 6.98 (d, 1H, J=8.8 Hz), 6.68 (s, 1H), 5.22 (s, 2H), 4.49 (d,1H, J=18.4 Hz), 3.84 (d, 1H, J=18.4 Hz).

3-[2-(4-Bromophenyl-2-oxoethyl)]-4,7-dichloro-3-hydroxy-1-1,3-dihydroindol-2-one(YK-4-283): light yellow solid; mp 200-202° C.; ¹H NMR (DMSO, 400 MHz) δ10.98 (s, 1H), 7.82 (d, 2H, J=8.8 Hz), 7.69 (d, 2H, J=8.8 Hz), 7.27 (d,1H, J=8.8 Hz), 6.87 (d, 1H, J=8.8 Hz), 6.45 (s, 1H), 4.32 (d, 1H, J=18.4Hz), 3.67 (d, 1H, J=18.0 Hz).

4,7-Dichloro-3-[2-(4-fluorophenyl-2-oxoethyl)]-3-hydroxyl-1,3-dihydroindol-2-one(YK-4-278): light yellow solid; mp 176-178° C.; ¹H NMR (DMSO, 400 MHz) δ10.98 (s, 1H), 7.98 (m, 2H), 7.29 (m, 3H), 6.87 (d, 1H, J=8.4 Hz), 6.43(s, 1H), 4.34 (d, 1H, J=18.4 Hz), 3.68 (d, 1H, J=18.4 Hz).

4,7-Dichloro-3-hydroxyl-3-[2-(4-iodophenyl-2-oxoethyl)]-1,3-dihydroindol-2-one(YK-4-277): white solid; mp 190-192° C.; ¹H NMR (DMSO, 400 MHz) δ 10.98(s, 1H), 7.87 (d, 2H, J=8.8 Hz), 7.64 (d, 2H, J=8.8 Hz), 7.27 (d, 1H,J=8.8 Hz), 6.87 (d, 1H, J=8.8 Hz), 6.44 (s, 1H), 4.30 (d, 1H, J=18.4Hz), 3.65 (d, 1H, J=18.4 Hz).

4,7-Dichloro-3-hydroxyl-3-(2-oxo-2-p-tolylethyl)-1,3-dihydroindol-2-one(YK-4-280): white solid; mp 189-192° C.; ¹H NMR (DMSO, 400 MHz) δ 10.95(s, 1H), 7.78 (d, 2H, J=8.4 Hz), 7.27 (m, 3H), 6.86 (d, 1H, J=8.8 Hz),6.41 (s, 1H), 4.33 (d, 1H, J=18.4 Hz), 3.64 (d, 1H, J=18.4 Hz), 2.33 (s,3H).

4,7-Dichloro-3-hydroxy-3-[2-(4-methoxyphenyl-2-oxoethyl)]-1,3-dihydroindol-2-one(YK-4-279): white solid; mp 149-151° C.; ¹H NMR (DMSO, 400 MH 1 z) δ10.93 (s, 1H), 7.86 (d, 2H, J=9.2 Hz), 7.26 (d, 1H, J=8.8 Hz), 6.98 (d,2H, J=8.8 Hz), 6.86 (d, 1H, J=8.4 Hz), 6.39 (s, 1H), 4.31 (d, 1H, J=18.0Hz), 3.80 (s, 3H), 3.61 (d, 1H, J=18.0 Hz).

4,7-Dichloro-3-hydroxyl-3-[2-(4-nitrophenyl-2-oxoethyl)]-1,3-dihydroindol-2-one(YK-4-286): yellow solid; mp 209-211° C.; ¹H NMR (DMSO, 400 MHz) δ 11.03(s, 1H), 8.27 (d, 2H, J=9.2 Hz), 8.13 (d, 2H, J=9.2 Hz), 7.28 (d, 1H,J=8.8 Hz), 6.88 (d, 1H, J=8.4 Hz), 6.51 (s, 1H), 4.38 (d, 1H, J=18.4Hz), 3.79 (d, 1H, J=18.0 Hz).

4,7-Dichloro-3-hydroxy-1-3-(2-oxo2-phenylethyl)-1,3-dihydroindol-2-one(YK-4-285): white solid; mp 198-200° C.; ¹H NMR (DMSO, 400 MHz) δ10.97(s, 1H), 7.88 (dd, 2H, J=0.8, 1.6 Hz), 7.62 (m, 1H), 7.48 (t, 2H, J=8.4,7.2 Hz), 7.27 (d, 1H, J=8.8 Hz), 6.87 (d, 1H, J=8.8 Hz), 6.43 (s, 1H),4.36 (d, 1H, J=18.4 Hz), 3.68 (d, 1H, J=18.4 Hz).

4,7-Dichloro-3-[2-(3-chlorophenyl-2-oxoethyl)]-3-hydroxyl-1,3-dihydroindol-2-one(YK-4-281): white solid; mp 201-203° C.; ¹H NMR (DMSO, 400 MHz) δ 11.01(s, 1H), 7.49 (m, 3H), 7.39 (m, 1H), 7.29 (d, 1H, J=8.4 Hz), 6.89 (d,1H, J=8.8 Hz), 6.49 (s, 1H), 4.17 (d, 1H, J=17.6 Hz), 3.64 (d, 1H,J=17.6 Hz).

4,7-Dichloro-3-[2-(2-chlorophenyl-2-oxoethyl)]-3-hydroxy-1-1,3-dihydroindol-2-one(YK-4-282): light yellow solid; mp 164-166° C.; ¹H NMR (DMSO, 400 MHz) δ11.00 (s, 1H), 7.87 (m, 2H), 7.69 (m, 1H), 7.52 (m, 1H), 7.28 (d, 1H,J=8.8 Hz), 6.87 (d, 1H, J=8.8 Hz), 6.45 (s, 1H), 4.33 (d, 1H, J=18.4Hz), 3.71 (d, 1H, J=18.4 Hz).

4,7-Dichloro-3-[2-(3,4-dichlorophenyl-2-oxoethyl)]-3-hydroxyl-1,3-dihydroindol-2-one(YK-4-287): white solid; mp 193-196° C.; ¹H NMR (DMSO, 400 MHz) δ 11.00(s, 1H), 8.09 (d, 1H, J=1.6 Hz), 7.86 (dd, 1H, J=1.6, 2.0 Hz), 7.75 (d,1H, J=8.4 Hz), 7.28 (d, 1H, J=8.8 Hz), 6.87 (d, 1H, J=8.4 Hz), 6.46 (s,1H), 4.32 (d, 1H, J=18.4 Hz), 3.72 (d, 1H, J=18.0 Hz).

4,7-Dichloro-3-hydroxy-1-3-[2-oxo-2-(2,3,4-trichlorophenylethyl)]-1,3-dihydroindol-2-one(YK-4-288): light yellow solid; mp 172-174° C.; ¹H NMR (DMSO, 400 MHz) δ11.03 (s, 1H), 7.71 (d, 1H, J=9.2 Hz), 7.48 (d, 1H, J=8.4 Hz), 7.29 (d,1H, J=8.8 Hz), 6.90 (d, 1H, J=8.8 Hz), 6.53 (s, 1H), 4.09 (d, 1H, J=16.4Hz), 3.66 (d, 1H, J=17.2 Hz).

4,7-Dichloro-3-hydroxy-1-3-[2-(4-hydroxyphenyl-2-oxoethyl)]-1,3-dihydroindol-2-one(YK-4-289): white solid; mp 240-242° C.; ¹H NMR (DMSO, 400 MHz) δ 10.91(s, 1H), 10.43 (br, 1H), 7.75 (d, 2H, J=8.8 Hz), 7.25 (d, 2H, J=8.8 Hz),6.85 (d, 1H, J=8.8 Hz), 6.78 (d, 1H, J=8.8 Hz), 6.36 (s, 1H), 4.27 (d,1H, J=18.0 Hz), 3.56 (d, 1H, J=17.6 Hz).

3-[2-(4-Aminophenyl-2-oxoethyl)]-4,7-dichloro-3-hydroxyl1,3-dihydroindol-2-one (YK-4-284): white solid; mp 240-243° C.; ¹H NMR(DMSO, 400 MHz) δ 10.85 (s, 1H), 7.56 (d, 2H, J=8.8 Hz), 7.24 (d, 1H,J=8.8 Hz), 6.84 (d, 1H, J=8.8 Hz), 6.49 (d, 2H, J=8.8 Hz), 6.28 (s, 1H),6.10 (s, 2H), 4.20 (d, 1H, J=18.0 Hz), 3.45 (d, 1H, J=17.6 Hz).

Depending upon the substituents present, the small molecule inhibitorscan be in a form of a pharmaceutically acceptable salt. The terms“pharmaceutically acceptable salt” as used herein are broad terms, andis to be given its ordinary and customary meaning to a person ofordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refers without limitation to salts preparedfrom pharmaceutically acceptable, non-toxic acids or bases. Suitablepharmaceutically acceptable salts include metallic salts, e.g., salts ofaluminum, zinc, alkali metal salts such as lithium, sodium, andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts; organic salts, e.g., salts of lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), procaine, and tris;salts of free acids and bases; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as, for example, The Merck Index. Anysuitable constituent can be selected to make a salt of the therapeuticagents discussed herein, provided that it is non-toxic and does notsubstantially interfere with the desired activity.

The compounds of preferred embodiments can include isomers, racemates,optical isomers, enantiomers, diastereomers, tautomers, and cis/transconformers. All such isomeric forms are included within preferredembodiments, including mixtures thereof. As discussed above, thecompounds of preferred embodiments may have chiral centers, for example,they may contain asymmetric carbon atoms and may thus exist in the formof enantiomers or diastereoisomers and mixtures thereof, e.g.,racemates. Asymmetric carbon atom(s) can be present in the (R)-, (S)-,or (R,S)-configuration, preferably in the (R)— or (S)— configuration, orcan be present as mixtures. Isomeric mixtures can be separated, asdesired, according to conventional methods to obtain pure isomers.

The compounds can be in amorphous form, or in crystalline forms. Thecrystalline forms of the compounds of preferred embodiments can exist aspolymorphs, which are included in preferred embodiments. In addition,some of the compounds of preferred embodiments may also form solvateswith water or other organic solvents. Such solvates are similarlyincluded within the scope of the preferred embodiments.

Peptide EWS-FLI1-Protein Inhibitors

Peptide EWS-FLI1-protein inhibitors preferably comprise the peptidesequence comprises SEQ ID NO: 29.

In some embodiments the peptide sequence comprises SEQ ID NO: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, and 31.

In some embodiments the peptide sequence comprises an N-terminal tagcontaining the cell-permeable Antennapedia peptide sequence (SEQ ID NO:34) linked to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31.

In certain embodiments the peptide sequence comprises an N-terminal tagcontaining a cell-penetrating cationic peptide sequence linked to SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31.

In certain embodiments the peptide comprises the sequence of a peptideisolated from a phage display library by the peptide's ability to bind aEWS-FLI1 protein. In some embodiments the peptide comprises the sequenceof a peptide isolated from a phage display library by the peptide'sability to bind a EWS-FLI1 protein homologue. In some embodiments thepeptide comprises the sequence of a peptide isolated from a phagedisplay library by the peptide's ability to bind a translocation fusionprotein. Preferred amino acid sequences for the peptides of preferredembodiments are provided in Table 1.

TABLE 1 SEQ ID Alternative NO: peptide IDs AMINO ACID SEQUENCE  1 A1TMRGKKKRTRAN  2 B12 QHRMASMSPTLP  3 D6 GLLPYRPREANF  4 D10 AMIPYTWFSPSP 5 D11 KQPKKAPRRIPQ  6 E5 SIPTTWFHPPPS  7 E6 GVSLHNTNWNIY  8 E7SDTSVNWLTLWY  9 E8 NTPQRPPYKRSP 28 E9 YTPPPLIEAFAT 10 F4 LAKSPSNSAREW 11F6 AKCHSDVPSPAC 12 F7 VHFKPTHLPSPP 13 F8 STSQALSRFPSF 14 F10GMMRALSHPSAS 15 F11 GTLTTPRLDLIM 16 G2 MKISAPALAFGL 17 G3 MFAKSPPYPSLM18 G4 FNWHWLSRPYFP 19 G5 FANHLTNAVHAL 20 G7 SQPWTNALVVSS 21 G8TAFWPLYPLSDW 22 G10 KLWNVPWPPHMR 23 G11 FTPPPAYGRNEG 24 H1 HWIPQTLPASFI25 H3 HHPFVTNTPSLI 26 H5 PNRLGRRPVRWE 27 H11 HWWYPLLPVRQM 29 E9RPPPLDAVIEA 30 P2A PAPLDAVIEA 31 D5A PPPLAAVIEA

Pharmaceutical Compositions

It is generally preferred to administer the inhibitors of preferredembodiments in an intravenous or subcutaneous unit dosage form; however,other routes of administration are also contemplated. Contemplatedroutes of administration include but are not limited to oral,parenteral, intravenous, and subcutaneous. The inhibitors of preferredembodiments can be formulated into liquid preparations for, e.g., oraladministration. Suitable forms include suspensions, syrups, elixirs, andthe like. Particularly preferred unit dosage forms for oraladministration include tablets and capsules. Unit dosage formsconfigured for administration once a day are particularly preferred;however, in certain embodiments it can be desirable to configure theunit dosage form for administration twice a day, or more.

The pharmaceutical compositions of preferred embodiments are preferablyisotonic with the blood or other body fluid of the recipient. Theisotonicity of the compositions can be attained using sodium tartrate,propylene glycol or other inorganic or organic solutes. Sodium chlorideis particularly preferred. Buffering agents can be employed, such asacetic acid and salts, citric acid and salts, boric acid and salts, andphosphoric acid and salts. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like.

Viscosity of the pharmaceutical compositions can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the thickening agentselected. An amount is preferably used that will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the pharmaceutical compositions. Benzyl alcohol can besuitable, although a variety of preservatives including, for example,parabens, thimerosal, chlorobutanol, or benzalkonium chloride can alsobe employed. A suitable concentration of the preservative is typicallyfrom about 0.02% to about 2% based on the total weight of thecomposition, although larger or smaller amounts can be desirabledepending upon the agent selected. Reducing agents, as described above,can be advantageously used to maintain good shelf life of theformulation.

The inhibitors of preferred embodiments can be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, or the like, and can contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,gelling or viscosity enhancing additives, preservatives, flavoringagents, colors, and the like, depending upon the route of administrationand the preparation desired. See, e.g., “Remington: The Science andPractice of Pharmacy”, Lippincott Williams & Wilkins; 20th edition (Jun.1, 2003) and “Remington's Pharmaceutical Sciences,” Mack Pub. Co.;18^(th) and 19^(th) editions (December 1985, and June 1990,respectively). Such preparations can include complexing agents, metalions, polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, and the like, liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. The presence of suchadditional components can influence the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance, andare thus chosen according to the intended application, such that thecharacteristics of the carrier are tailored to the selected route ofadministration.

For oral administration, the pharmaceutical compositions can be providedas a tablet, aqueous or oil suspension, dispersible powder or granule,emulsion, hard or soft capsule, syrup or elixir. Compositions intendedfor oral use can be prepared according to any method known in the artfor the manufacture of pharmaceutical compositions and can include oneor more of the following agents: sweeteners, flavoring agents, coloringagents and preservatives. Aqueous suspensions can contain the activeingredient in admixture with excipients suitable for the manufacture ofaqueous suspensions.

Formulations for oral use can also be provided as hard gelatin capsules,wherein the active ingredient(s) are mixed with an inert solid diluent,such as calcium carbonate, calcium phosphate, or kaolin, or as softgelatin capsules. In soft capsules, the inhibitors can be dissolved orsuspended in suitable liquids, such as water or an oil medium, such aspeanut oil, olive oil, fatty oils, liquid paraffin, or liquidpolyethylene glycols. Stabilizers and microspheres formulated for oraladministration can also be used. Capsules can include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredient in admixture with fillers such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers.

Tablets can be uncoated or coated by known methods to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period of time. For example, atime delay material such as glyceryl monostearate can be used. Whenadministered in solid form, such as tablet form, the solid formtypically comprises from about 0.001 wt. % or less to about 50 wt. % ormore of active ingredient(s), preferably from about 0.005, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, or 45 wt. %.

Tablets can contain the active ingredients in admixture with non-toxicpharmaceutically acceptable excipients including inert materials. Forexample, a tablet can be prepared by compression or molding, optionally,with one or more additional ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredients ina free-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered inhibitor moistened with an inert liquid diluent.

Preferably, each tablet or capsule contains from about 1 mg or less toabout 1,000 mg or more of an inhibitor of the preferred embodiments,more preferably from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mgto about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, or 900 mg. Most preferably, tablets or capsules are providedin a range of dosages to permit divided dosages to be administered. Adosage appropriate to the patient and the number of doses to beadministered daily can thus be conveniently selected. In certainembodiments it can be preferred to incorporate two or more of thetherapeutic agents to be administered into a single tablet or otherdosage form (e.g., in a combination therapy); however, in otherembodiments it can be preferred to provide the therapeutic agents inseparate dosage forms.

Suitable inert materials include diluents, such as carbohydrates,mannitol, lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans, starch, and the like, or inorganic salts such as calciumtriphosphate, calcium phosphate, sodium phosphate, calcium carbonate,sodium carbonate, magnesium carbonate, and sodium chloride.Disintegrants or granulating agents can be included in the formulation,for example, starches such as corn starch, alginic acid, sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite, insoluble cationic exchange resins,powdered gums such as agar, karaya or tragacanth, or alginic acid orsalts thereof.

Binders can be used to form a hard tablet. Binders include materialsfrom natural products such as acacia, tragacanth, starch and gelatin,methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, and the like.

Lubricants, such as stearic acid or magnesium or calcium salts thereof,polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes,sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol,starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like,can be included in tablet formulations.

Surfactants can also be employed, for example, anionic detergents suchas sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctylsodium sulfonate, cationic such as benzalkonium chloride or benzethoniumchloride, or nonionic detergents such as polyoxyethylene hydrogenatedcastor oil, glycerol monostearate, polysorbates, sucrose fatty acidester, methyl cellulose, or carboxymethyl cellulose.

Controlled release formulations can be employed wherein the amifostineor analog(s) thereof is incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms. Slowly degeneratingmatrices can also be incorporated into the formulation. Other deliverysystems can include timed release, delayed release, or sustained releasedelivery systems.

Coatings can be used, for example, nonenteric materials such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethylcellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose,sodium carboxy-methyl cellulose, providone and the polyethylene glycols,or enteric materials such as phthalic acid esters. Dyestuffs or pigmentscan be added for identification or to characterize differentcombinations of inhibitor doses

When administered orally in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils can be added to theactive ingredient(s). Physiological saline solution, dextrose, or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol, or polyethylene glycol are also suitable liquid carriers. Thepharmaceutical compositions can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil, such as olive orarachis oil, a mineral oil such as liquid paraffin, or a mixturethereof. Suitable emulsifying agents include naturally-occurring gumssuch as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsions can also containsweetening and flavoring agents.

Pulmonary delivery of the inhibitor can also be employed. The inhibitoris delivered to the lungs while inhaling and traverses across the lungepithelial lining to the blood stream. A wide range of mechanicaldevices designed for pulmonary delivery of therapeutic products can beemployed, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. These devices employ formulations suitable for thedispensing of inhibitor. Typically, each formulation is specific to thetype of device employed and can involve the use of an appropriatepropellant material, in addition to diluents, adjuvants, and/or carriersuseful in therapy.

The inhibitor and/or other optional active ingredients areadvantageously prepared for pulmonary delivery in particulate form withan average particle size of from 0.1 μm or less to 10 μm or more, morepreferably from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μm toabout 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, or 9.5 μm. Pharmaceutically acceptable carriers forpulmonary delivery of inhibitor include carbohydrates such as trehalose,mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients foruse in formulations can include DPPC, DOPE, DSPC, and DOPC. Natural orsynthetic surfactants can be used, including polyethylene glycol anddextrans, such as cyclodextran. Bile salts and other related enhancers,as well as cellulose and cellulose derivatives, and amino acids can alsobe used. Liposomes, microcapsules, microspheres, inclusion complexes,and other types of carriers can also be employed.

Pharmaceutical formulations suitable for use with a nebulizer, eitherjet or ultrasonic, typically comprise the inhibitor dissolved orsuspended in water at a concentration of about 0.01 or less to 100 mg ormore of inhibitor per mL of solution, preferably from about 0.1, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulationcan also include a buffer and a simple sugar (e.g., for proteinstabilization and regulation of osmotic pressure). The nebulizerformulation can also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the inhibitor caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the active ingredientssuspended in a propellant with the aid of a surfactant. The propellantcan include conventional propellants, such as chlorofluorocarbons,hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons.Preferred propellants include trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol,1,1,1,2-tetrafluoroethane, and combinations thereof. Suitablesurfactants include sorbitan trioleate, soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typicallycomprise a finely divided dry powder containing inhibitor, optionallyincluding a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in an amount that facilitates dispersal of thepowder from the device, typically from about 1 wt. % or less to 99 wt. %or more of the formulation, preferably from about 5, 10, 15, 20, 25, 30,35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. %of the formulation.

When an inhibitor of the preferred embodiments is administered byintravenous, parenteral, or other injection, it is preferably in theform of a pyrogen-free, parenterally acceptable aqueous solution oroleaginous suspension. Suspensions can be formulated according tomethods well known in the art using suitable dispersing or wettingagents and suspending agents. The preparation of acceptable aqueoussolutions with suitable pH, isotonicity, stability, and the like, iswithin the skill in the art. A preferred pharmaceutical composition forinjection preferably contains an isotonic vehicle such as1,3-butanediol, water, isotonic sodium chloride solution, Ringer'ssolution, dextrose solution, dextrose and sodium chloride solution,lactated Ringer's solution, or other vehicles as are known in the art.In addition, sterile fixed oils can be employed conventionally as asolvent or suspending medium. For this purpose, any bland fixed oil canbe employed including synthetic mono or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the formation ofinjectable preparations. The pharmaceutical compositions can alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

The duration of the injection can be adjusted depending upon variousfactors, and can comprise a single injection administered over thecourse of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours or more of continuous intravenous administration.

The inhibitors of the preferred embodiments can additionally employadjunct components conventionally found in pharmaceutical compositionsin their art-established fashion and at their art-established levels.Thus, for example, the compositions can contain additional compatiblepharmaceutically active materials for combination therapy (such assupplementary antimicrobials, antipruritics, astringents, localanesthetics, anti-inflammatory agents, reducing agents,chemotherapeutics and the like), or can contain materials useful inphysically formulating various dosage forms of the preferredembodiments, such as excipients, dyes, thickening agents, stabilizers,preservatives or antioxidants. Anti-cancer agents that can be used incombination with the inhibitors of preferred embodiments include, butare not limited to, vinca alkaloids such as vinblastine and vincristine;anthracyclines such as doxorubicin, daunorubicin, epirubicin;anthracenes such as bisantrene and mitoxantrone; epipodophyllo-toxinssuch as etoposide and teniposide; and other anticancer drugs such asactinomyocin D, mithomycin C, mitramycin, methotrexate, docetaxel,etoposide (VP-16), paclitaxel, docetaxel, and adriamycin); andimmunosuppressants (e.g., cyclosporine A, tacrolimus).

The inhibitors of the preferred embodiments can be provided to anadministering physician or other health care professional in the form ofa kit. The kit is a package which houses a container which contains theinhibitor(s) in a suitable pharmaceutical composition, and instructionsfor administering the pharmaceutical composition to a subject. The kitcan optionally also contain one or more additional therapeutic agents,e.g., chemotherapeutics currently employed for treating the sarcomasdescribed herein. For example, a kit containing one or more compositionscomprising inhibitor(s) of the preferred embodiments in combination withone or more additional chemotherapeutic agents can be provided, orseparate pharmaceutical compositions containing an inhibitor of thepreferred embodiments and additional therapeutic agents can be provided.The kit can also contain separate doses of an inhibitor of the preferredembodiments for serial or sequential administration. The kit canoptionally contain one or more diagnostic tools and instructions foruse. The kit can contain suitable delivery devices, e.g., syringes, andthe like, along with instructions for administering the inhibitor(s) andany other therapeutic agent. The kit can optionally contain instructionsfor storage, reconstitution (if applicable), and administration of anyor all therapeutic agents included. The kits can include a plurality ofcontainers reflecting the number of administrations to be given to asubject.

Mechanism of Action

Ewing's sarcoma family of tumors (ESFT) contains the unique FusionProtein EWS-FLI1. ESFT affects patients between the ages of 3 and 40years, with most cases occurring in the second decade. Although theembryologic cell type from which ESFT are derived is unknown, the tumoroften grows in close proximity to bone, but can occur as a soft-tissuemass. Over 40% of patients who present with localized tumors willdevelop recurrent disease and the majority of these will die from ESFT,while 75-80% of patients who present with metastatic ESFT will diewithin 5 years despite high-dose chemotherapy (Grier H E, Krailo M D,Tarbell N J, et al. Addition of ifosfamide and etoposide to standardchemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor ofbone. N Engl J Med 2003; 348(8):694-701). These survival rates have notimproved for the past 20 years, even after dose-intensifyingchemotherapy. To improve survival and reduce therapy-related morbidity,novel targeted strategies for treating ESFT patients, as provided in thepreferred embodiments, can be employed. ESFT are characterized by atranslocation, occurring in 95% of tumors, between the central exons ofthe EWS gene (Ewing Sarcoma) located on chromosome 22 to the centralexons of an ets family gene; either FLI1 (Friend Leukemia Insertion)located on chromosome 11, t(11; 22), or ERG located on chromosome 21,t(21; 22). The EWS-FLI1 fusion transcript encodes a 55 kDa protein(electrophoretic motility of approximately 68 kD) with two primarydomains. The EWS domain is a potent transcriptional activator, while theFLI1 domain contains a highly conserved ets DNA binding domain (May W A,Lessnick S L, Braun B S, et al. The Ewing's sarcoma EWS/FLI-1 fusiongene encodes a more potent transcriptional activator and is a morepowerful transforming gene than FLI-1. Mol Cell Biol 1993;13(12):7393-8); the resulting EWS-FLI1 fusion protein acts as anaberrant transcription factor. EWS-FLI1 transformation of mousefibroblasts requires both the EWS and FLI1 functional domains to beintact (May W A, Gishizky M L, Lessnick S L, et al. Ewing sarcoma 11; 22translocation produces a chimeric transcription factor that requires theDNA-binding domain encoded by FLI1 for transformation. Proc Natl AcadSci USA 1993; 90(12):5752-6).

EWS-FLI1 is an outstanding therapeutic target, in that it is expressedonly in tumor cells and is required to maintain the growth of ESFT celllines. Reduced expression levels of EWS-FLI1 using either antisenseoligodeoxynucleotides (ODN) (Toretsky J A, Connell Y, Neckers L, Bhat NK. Inhibition of EWS-FLI-1 fusion protein with antisenseoligodeoxynucleotides. J Neurooncol 1997; 31 (1-2):9-16; Tanaka K,Iwakuma T, Harimaya K, Sato H, Iwamoto Y. EWS-Flit antisenseoligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma andprimitive neuroectodermal tumor cells. J Clin Invest 1997; 99(2):239-47)or small interfering RNAs (siRNA) (Ouchida M, Ohno T, Fujimura Y, Rao VN, Reddy E S. Loss of tumorigenicity of Ewing's sarcoma cells expressingantisense RNA to EWS-fusion transcripts. Oncogene 1995; 11(6):1049-54;Maksimenko A, Malvy C, Lambert G, et al. Oligonucleotides targetedagainst a junction oncogene are made efficient by nanotechnologies.Pharm Res 2003; 20(10):1565-7; Kovar H, Aryee D N, Jug G, et al.EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells invitro. Cell Growth Differ 1996; 7(4):429-37) cause decreasedproliferation of ESFT cell lines and regression of tumors in nude mice.Recent advances in nanotechnology have improved the delivery andcontrolled release of siRNA, yet neither antisense ODN nor siRNAreduction of EWS-FLI1 in humans is possible with current technologies(Maksimenko A, Malvy C, Lambert G, et al. Oligonucleotides targetedagainst a junction oncogene are made efficient by nanotechnologies.Pharm Res 2003; 20(10):1565-7; Lambert G, Bertrand J R, Fattal E, et al.EWS fli-1 antisense nanocapsules inhibits Ewing sarcoma-related tumor inmice. Biochem Biophys Res Commun 2000; 279(2):401-6). One interestingapproach to EWS-FLI1 targeting used comparative expression between siRNAreduced EWS-FLI1 and a library of small molecules, which led to acurrent clinical trial with Ara-C (Stegmaier K, Wong J S, Ross K N, etal. Signature-based small molecule screening identifies cytosinearabinoside as an EWS/FLI modulator in Ewing sarcoma. PLoS medicine2007; 4 (4):e122). This method of identifying Ara-C also indicateddoxorubicin and puromycin would reduce EWS-FLI1 levels. Doxorubicin iscurrently used as standard therapy for ESFT patients and yet, survivalis far from acceptable (Grier H E, Krailo M D, Tarbell N J, et al.Addition of ifosfamide and etoposide to standard chemotherapy forEwing's sarcoma and primitive neuroectodermal tumor of bone. N Engl JMed 2003; 348(8):694-701). The use of Ara-C in ESFT patients iscurrently being evaluated in a Phase II trial. While it is hoped thatthis represents a needed clinical breakthrough, it certainlydemonstrates the importance of small molecule targeting of EWS-FLI1. Thepreferred embodiments provide small molecule protein-protein interactioninhibitors (SMPPII) that disrupt EWS-FLI1 from critical proteinpartners, thereby achieving tumor specificity and more precise targetingof EWS-FLI1.

There is sufficient evidence to conclude that EWS-FLI1 fusion proteinfunctions differently than either untranslocated EWS or FLI1 (May W A,Gishizky M L, Lessnick S L, et al. Ewing sarcoma 11; 22 translocationproduces a chimeric transcription factor that requires the DNA-bindingdomain encoded by FLI1 for transformation. Proc Natl Acad Sci USA 1993;90(12):5752-6). Changes in gene expression profiles ofEWS-FLI1-expressing cell lines (Braun B S, Frieden R, Lessnick S L, MayW A, Denny C T. Identification of target genes for the Ewing's sarcomaEWS/FLI fusion protein by representational difference analysis. Mol CellBiol 1995; 15(8):4623-30) or tumor cells taken from ESFT patients,compared to tumors lacking EWS-FLI1 expression, indicate that EWS-FLI1may play a role in transcriptional regulation (Khan J, Wei J S, RingnerM, et al. Classification and diagnostic prediction of cancers using geneexpression profiling and artificial neural networks. Nat Med 2001;7(6):673-9; Baer C, Nees M, Breit S, et al. Profiling and functionalannotation of mRNA gene expression in pediatric rhabdomyosarcoma andEwing's sarcoma. Int J Cancer 2004; 110(5):687-94). While a clearpicture of the mechanism of EWS-FLI1-regulated gene expression has yetto emerge, this activity is likely the result of direct or secondaryinteractions between EWS-FLI1 and regulators of RNA synthesis andsplicing (Uren A, Toretsky J A. Ewing's Sarcoma Oncoprotein EWS-FLI1:the Perfect Target without a Therapeutic Agent. Future One 2005;1(4):521-8).

EWS-FLI1 is a great therapeutic target since it is only expressed intumor cells; however, the ability to target this tumor-specific oncogenehas previously not been successful. One of the challenges towards smallmolecule development is that EWS-FLI1 lacks any know enzymatic domains,and enzyme domains have been thought to be critical for targetedtherapeutics. In addition, EWS-FLI1 is a disordered protein, indicatingthat it does not exhibit a rigid structure that can be used forstructure based drug design (Uren A, Tcherkasskaya O, Toretsky J A.Recombinant EWS-FLI1 oncoprotein activates transcription. Biochemistry2004; 43(42):13579-89). In fact, the disordered nature of EWS-FLI1 iscritical for its transcriptional regulation (Ng K P, Potikyan G, SaveneR O, Denny C T, Uversky V N, Lee K A. Multiple aromatic side chainswithin a disordered structure are critical for transcription andtransforming activity of EWS family oncoproteins. Proc Natl Acad Sci U SA 2007; 104(2):479-84). Disordered proteins are considered as moreattractive targets for small molecule protein-protein interactioninhibitors specifically because of their biochemical disorderedproperties (Cheng Y, LeGall T, Oldfield C J, et al. Rational drug designvia intrinsically disordered protein. Trends Biotechnol 2006;24(10):435-42).

EWS-FLI1 binds RNA helicase A in vitro and in vivo. It is believed thatprotein-protein interactions of EWS-FLI1 may contribute to its oncogenicpotential; therefore, novel proteins have been sought that directlyinteract with and functionally modulate EWS-FLI1. Recombinant EWS-FLI1that is transcriptionally active (Uren A, Tcherkasskaya O, Toretsky J A.Recombinant EWS-FLI1 oncoprotein activates transcription. Biochemistry2004; 43(42):13579-89) was used as a target for screening a commercialpeptide phage display library. Twenty-eight novel peptides thatdifferentially bind to EWS-FLI1 were identified from phage sequencing. ANational Center for Biotechnology Information database search for humanproteins homologous to these peptides identified a peptide that washomologous to aa 823-832 of the human RNA helicase A, (RHA, gene bankaccession number A47363) (Toretsky J A, Erkizan V, Levenson A, et al.Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res2006; 66(11):5574-81).

RHA, a member of the highly conserved DEXD/H box helicase family ofproteins, is an integral, multifunctional member of the humantranscriptome (Zhang S, Grosse F. Multiple functions of nuclear DNAhelicase II (RNA helicase A) in nucleic acid metabolism. Acta BiochimBiophys Sin(Shanghai) 2004; 36(3):177-83; von Hippel P H, Delagoutte E.A general model for nucleic acid helicases and their “coupling” withinmacromolecular machines. Cell 2001; 104(2):177-90). These proteins areinvolved in diverse functions in a variety of organisms, from archaea,eubacteria, lower and higher eukaryotes and a number of viruses,including the positive-sense RNA viruses of the Flavivirus family. RHAis a transcriptional coactivator for NF-κB, and has been shown to formcomplexes with Creb-binding protein (CBP) (Nakajima T, Uchida C,Anderson S F, et al. RNA helicase A mediates association of CBP with RNApolymerase II. Cell 1997; 90(6):1107-12), RNA Polymerase II (Nakajima T,Uchida C, Anderson S F, et al. RNA helicase A mediates association ofCBP with RNA polymerase II. Cell 1997; 90(6):1107-12), the breast cancertumor suppressor BRCA1 (Anderson S F, Schlegel B P, Nakajima T, Wolpin ES, Parvin J D. BRCA1 protein is linked to the RNA polymerase IIholoenzyme complex via RNA helicase A. Nat Genet. 1998; 19(3):254-6),and, most recently, EWS-FLI1 (Toretsky J A, Erkizan V, Levenson A, etal. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. CancerRes 2006; 66(11):5574-81). EWS-FLI1 binds to a region of RHA that isunique and not known as a binding site for any of the other RHA bindingpartners (Toretsky J A, Erkizan V, Levenson A, et al. OncoproteinEWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 2006;66(11):5574-81). RHA expression enhanced EWS-FLI1 mediatedanchorage-independent colony formation, while an inactivating mutationof RHA prevented colony formation (Toretsky J A, Erkizan V, Levenson A,et al. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A.Cancer Res 2006; 66(11):5574-81). This structural and functioninteraction is the basis for the therapeutic agents of preferredembodiments.

Despite the importance of transcription in tumorigenesis, the role ofhelicases in this process has not been well-studied. RHA is an integralmember of the human transcriptome with diverse functions (Zhang S,Grosse F. Multiple functions of nuclear DNA helicase II (RNA helicase A)in nucleic acid metabolism. Acta Biochim Biophys Sin (Shanghai) 2004;36(3):177-83; von Hippel P H, Delagoutte E. A general model for nucleicacid helicases and their “coupling” within macromolecular machines. Cell2001; 104(2):177-90). Our recently published data show that RHAinteracts with the multifunctional EWS-FLI1 oncoprotein (Toretsky J A,Erkizan V, Levenson A, et al. Oncoprotein EWS-FLI1 activity is enhancedby RNA helicase A. Cancer Res 2006; 66(11):5574-81). This interactioncould account for the observed ability of EWS-FLI1 to function in bothtranscription initiation and post-transcriptional RNA modification. RNAhelicases are also known to bind and act as a bridge for some of thesame factors that have been identified as binding partners for EWS-FLI1,including the splicing factor U1C (Chen J Y, Stands L, Staley J P,Jackups R R, Jr., Latus L J, Chang T H. Specific alterations of U1-Cprotein or U1 small nuclear RNA can eliminate the requirement of Prp28p,an essential DEAD box splicing factor. Mol Cell 2001; 7(1):227-32; KnoopL L, Baker S J. The splicing factor U1C represses EWS/FLI-mediatedtransactivation. J Biol Chem 2000; 275(32):24865-71), Creb-bindingprotein (CBP) (Nakajima T, Uchida C, Anderson S F, et al. RNA helicase Amediates association of CBP with RNA polymerase II. Cell 1997;90(6):1107-12) and RNA Polymerase II (Nakajima T, Uchida C, Anderson SF, et al. RNA helicase A mediates association of CBP with RNA polymeraseII. Cell 1997; 90(6):1107-12). RHA may perform a similar function forEWS-FLI1 and RNA Pol II, acting in the recruitment of key processingproteins. RHA may also contribute to ESFT oncogenesis by maintainingEWS-FLI1 as part of a large transcriptional complex whose functionrelies on the ATPase activity of RHA as an energy source. Finally,helicases, like RHA, can stabilize mRNA species (Iost I, Dreyfus M.mRNAs can be stabilized by DEAD-box proteins. Nature 1994;372(6502):193-6). The stabilization and metabolism of EWS-FLI1transcribed mRNA by RHA may augment the oncogenic nature of EWS-FLI1.

While EWS-FLI1 is quite specific to ESFT cells, EWS and RHA areubiquitously expressed. The region between EWS-FLI1 and RHA are targetedby molecular therapeutics that may have specificity; since EWS-FLI1 isexpressed only in tumors and the interaction points with RHA may beunique. Two different types of therapeutic agents are provided toinhibit EWS-FLI1 function, peptide inhibitors and small moleculesprotein-protein interaction inhibitors (SMPPII).

Peptides that inhibit protein-protein interactions have been developedas molecular therapeutics because of their specificity of interaction.While there are challenges in the delivery of these peptides, theirspecificity allows for their development in laboratory models andproof-of-principle experiments. The AF4-MLL fusion protein in acuteleukemia has been targeted by peptides and leads to cell death (PalermoC M, Bennett C A, Winters A C, Hemenway C S. The AF4-mimetic peptide,PFWT, induces necrotic cell death in MV4-11 leukemia cells. Leuk Res2007; Srinivasan R S, Nesbit J B, Marrero L, Erfurth F, LaRussa V F,Hemenway C S. The synthetic peptide PFWT disrupts AF4-AF9 proteincomplexes and induces apoptosis in t(4; 11) leukemia cells. Leukemia2004; 18(8):1364-72). Another peptide prevents Hsp90 from stabilizingchaperone partners and reduces mouse model xenografts of multiple tumors(Gyurkocza B, Plescia J, Raskett C M, et al. Antileukemic activity ofshepherdin and molecular diversity of hsp90 inhibitors. J Natl CancerInst 2006; 98(15):1068-77; Plescia J, Salz W, Xia F, et al. Rationaldesign of shepherdin, a novel anticancer agent. Cancer Cell 2005;7(5):457-68). Activation of p53 enhanced the survival in mice withperitoneal carcinomatosis (Snyder E L, Meade B R, Saenz C C, Dowdy S F.Treatment of terminal peritoneal carcinomatosis by a transduciblep53-activating peptide. PLoS Biol 2004; 2 (2):E36). Naturally occurringand synthetic defensin peptides are important as antimicrobial andimmune modulation agents (Bowdish D M, Davidson D J, Hancock R E.Immunomodulatory properties of defensins and cathelicidins. Curr TopMicrobiol Immunol 2006; 306:27-66). Peptides are now being evaluated forreducing organ graft rejection by immune modulation (Lang J, Zhan J, XuL, Yan Z. Identification of peptide mimetics of xenoreactive alpha-Galantigenic epitope by phage display. Biochem Biophys Res Commun 2006;344(1):214-20). A biologically active inhibitory peptide sequence (E9RP)with 4 μM binding affinity to EWS-FLI1 that inhibits ESFT cell growthand can be delivered to cell culture, expressed in cells, or injectedinto animals has been developed.

Disruption of protein-protein interactions is thought to be a verychallenging, albeit surmountable, target for small moleculestherapeutics (Sillerud L O, Larson R S. Design and structure of peptideand peptidomimetic antagonists of protein-protein interaction. CurrProtein Pept Sci 2005; 6(2):151-69; Pagliaro L, Felding J, Audouze K, etal. Emerging classes of protein-protein interaction inhibitors and newtools for their development. Curr Opin Chem Biol 2004; 8(4):442-9).Recently, more investigators are both exploring the methodology ofidentifying small molecule protein-protein interaction inhibitors(SMPPII) (Murray J K, Gellman S H. Targeting protein-proteininteractions: Lessons from p53/MDM2. Biopolymers 2007; 88(5):657-86). Inaddition, some early results supports the feasibility of inhibiting thefrizzled receptor from downstream modulator disheveled (Fujii N, You L,Xu Z, et al. An antagonist of disheveled protein-protein interactionsuppresses beta-catenin-dependent tumor cell growth. Cancer Res 2007;67(2):573-9). The vinca alkaloids represent a class of small moleculecontaining extremely effective anti-cancer agents that exert theireffects by binding to β-tubulin and inhibiting its polymerization (GadekT R, Nicholas J B. Small molecule antagonists of proteins. BiochemPharmacol 2003; 65(1):1-8). Given the significant challenges of systemicoligonucleotide delivery in patients, and the very successful transportof small molecule pharmacologic agents, drug discovery efforts have beendirected towards identification of SMPPII. Two independent groups havedeveloped SMPPII of the myc:max heterodimer based upon library screening(Berg T, Cohen S B, Desharnais J, et al. Small-molecule antagonists ofMyc/Max dimerization inhibit Myc-induced transformation of chickenembryo fibroblasts. Proc Natl Acad Sci USA 2002; 99(6):3830-5; Yin X,Giap C, Lazo J S, Prochownik E V. Low molecular weight inhibitors ofMyc-Max interaction and function. Oncogene 2003; 22(40): 6151-9).

Most translocation-fusion protein sarcomas portend a poor prognosis,including ESFT. The chromosomal translocation t(11; 22), leading to theunique and critical fusion protein EWS-FLI1, is a perfect cancer target.Many other sarcomas share similar translocation variants (Table 2. fromHelman L J, Meltzer P. Mechanisms of sarcoma development. Nat Rev Cancer2003; 3(9):685-94).

EWS-FLI1 translocations have been reported in solidpseudopapillaryneoplasms of the pancreas (Maitra A., et al., Detectionof t(11; 22)(q24; q12) translocation and EWS-FLI-1 fusion transcript ina case of solid pseudopapillary tumor of the pancreas. Pediatr DevPathol 2000; 3:603-605), however the role of EWS-FLI1 in all solidpseudopaillary neoplasms remains to be resolved (Katharina Tiemann etal., Solid pseudopapillary neoplasms of the pancreas are associated withFLI-1 expression, but not with EWS/FLI-1 translocation).

EWS or FLI1 homologues are partners in translocations that occur in awide range of sarcomas and leukemias. EWS, or its homologue TLS or FUS,is involved in chromosomal translocations of clear cell sarcoma, myxoidliposarcoma, desmoplastic small round cell tumor, chondrosarcoma andacute myeloid leukemia. FLI1 belongs to the ets family of genes. TheFLI1 homologue ERG is translocated in approximately 10% of Ewing'ssarcomas and 20% of acute myeloid leukemias. This suggests that EWS-FLI1can serve as model system that might impact upon a family of diseases(related by translocation partners) that affect a large number ofpatients (Uren A., Tcherkasskaya O. and Toretsky J. A. RecombinantEWS-FLI1 oncoprotein activates transcription. Biochemistry 43 (42)13579-89 (2004)).

ERG is also translocated in prostate cancer, where the TMPRSS2:ERGfusion suggests a distinct molecular subtype that may define risk fordisease progression (F. Demichelis et al., TMPRSS2:ERG gene fusionassociated with lethal cancer in a watchful waiting cohort. Oncogene(2007) 26, 4596-4599). Other diseases where translocations of EWS orFLI1 family members have been observed include congenital fibrosarcomaand cellular mesobalstic nephroma where the ets family member ETV6 isjuxtaposed with NTRK3. Other translocation gene fusions include chronicmyeloid leukemia that leads to expression of the BCR-ABL fusion protein,and synovial sarcoma where the SYT gene from chromosome 18 is juxtaposedwith either SSX1 or SSX2 from the X chromosome (Aykut Uren and JeffreyA. Toretsky, Pediatric malignancies provide unique cancer therapytargets. Curr Opin Pediatr 17:14-19 (2005)).

Therefore, the therapeutic agents of the preferred embodiments havepotential for application in many other tumors. More broadly, some ofthe most difficult leukemias also have translocation-generated fusionproteins involving the mixed-lineage leukemia gene (MLL, 11q23), and ourwork could serve as a paradigm for a very treatment-resistant group ofcancers (Pui C H, Chessells J M, Camitta B, et al. Clinicalheterogeneity in childhood acute lymphoblastic leukemia with 11q23rearrangements. Leukemia 2003; 17(4):700-6.). Thus embodiments includecancers where translocations have occurred. Translocation fusion genesare listed in Table 2.

TABLE 2 Translocation Fusion-Genes in Sarcoma Translocation Genes Typeof fusion gene Ewing's sarcoma t(11; 22)(q24; q12) EWSR1-FLI1Transcription factor t(21; 22)(q22; q12) EWSR1-ERG Transcription factort(7; 22)(p22; q12) EWSR1-ETV1 Transcription factor t(17; 22)(q21; q12)EWSR1-ETV4 Transcription factor t(2; 22)(q33; q12) EWSR1-FEVTranscription factor Clear-cell sarcoma t(12; 22)(q13; q12) EWSR1-ATF1Transcription factor Desmoplastic small round-cell tumor t(11; 22)(p13:q12) EWSR1-WT1 Transcription factor Myxoid chondrosarcoma t(9;22)(q22-31; q11-12) EWSR1-NR4A3 Transcription factor Myxoid liposarcomat(12; 16)(q13; p11) FUS-DDIT3 Transcription factor t(12; 22)(q13; q12)EWSR1-DDIT3 Transcription factor Alveolar rhabdomyosarcoma t(2; 13)(q35;q14) PAX3-FOXO1A Transcription factor t(1; 13)(p36; q14) PAX7-FOXO1ATranscription factor Synovial sarcoma t(X; 18)(p11; q11) SYT-SSXTranscription factor Dermatofibrosarcoma protuberans t(17; 22)(q22; q13)COL1A1-PDGFB Growth factor Congenital fibrosarcoma t(12; 15)(p13; q25)ETV6-NTRK3 Transcription-factor receptor Inflammatory myofibroblastictumor 2p23 rearrangements TMP3-ALK; Growth-factor receptor TMP4-ALKAlveolar soft-part sarcoma t(X; 17)(p11.2; q25) ASPL-TFE3 Transcriptionfactor

Experimental

Experiments have been conducted validating the functional importance ofthe interaction and demonstrating how SPR can identify small moleculesthat bind to EWS-FLI1. These experiments include a demonstration ofdirect RHA binding to EWS-FLI1, RHA required for optimal EWS-FLI1function, a peptide that binds to EWS-FLI1, blocks RHA binding andreduces tumorigenesis, small molecule screening and compoundidentification, and optimization and synthetic strategies for developingtherapeutic compounds.

EWS-FLI1 Binds to a Unique Region of RHA

RNA Helicase A (RHA) has been established as a partner of EWS-FLI1(Toretsky J A, Erkizan V, Levenson A, et al. Oncoprotein EWS-FLI1activity is enhanced by RNA helicase A. Cancer Res 2006;66(11):5574-81). In order to show that RHA was directly binding toEWS-FLI1, ELISA and solution immunoprecipitation assays were developed.ELISA assay suggested the direct binding of EWS-FLI1 to RHA (FIG. 1A).RHA protein specifically bound EWS-FLI1 compared with bovine serumalbumin (BSA) in a dose-dependent manner (FIG. 1B).

ELISA was used to identify the region of RHA that binds to EWS-FLI1.GST-tagged human RHA fragments aa 1-88, 1-250, 230-325, 326-650,630-1020 and 1000-1279 (Anderson S F, Schlegel B P, Nakajima T, Wolpin ES, Parvin J D. BRCA1 protein is linked to the RNA polymerase IIholoenzyme complex via RNA helicase A. Nat Genet. 1998; 19(3):254-6)were analyzed for EWS-FLI1 binding in an ELISA assay normalized usingbovine serum albumin (BSA) (FIG. 2A). Results showed that GST-RHA(630-1020) bound to EWS-FLI1 14-fold greater than it bound to BSA.GST-RHA (630-1020) contains homology (aa 823-832) to the E9R peptidesequence (later used to develop inhibitory peptides). Fragment GST-RHA(630-1020) also bound to endogenous EWS-FLI1, in a GST pull-downimmunoprecipitation assay using endogenous ESFT cell lysate followed byFLI1 antibody western blot (FIG. 2C). The immunoprecipitation studyidentified a potential second site of interaction in the C-terminal RHA(1000-1279). This potential second site of interaction will be evaluatedin other proposals. This data enabled a robust, reproducible assay toevaluate RHA binding to EWS-FLI1 to be created.

RHA Binding to EWS-FLI1 is Required for Optimal Soft-Agar Colony Growth

GST-RHA (630-1020) had the strongest evidence for containing asignificant binding site to EWS-FLI1 including containing the phagedisplay peptide that identified RHA as a putative EWS-FLI1 partner aswell as immunoprecipitation and ELISA assays. The amino-acids from theregion of GST-RHA (630-1020) were independently mutated and mutants thatdid not bind to EWS-FLI1 were identified. These EWS-FLI1 “non-binding”mutants were prepared in the full length RHA expression plasmid.

Using the standard anchorage-independent growth assay with primarymurine embryonic fibroblasts (containing a small amount of endogenousRHA), expression of EWS-FLI1 induced colony formation as didover-expression of the wild-type RHA (FIG. 3, open bars). When the twoproteins were co-expressed there was a synergistic increase in colonyformation (FIG. 3, orange bar). However, when RHA D827A, a mutant thatdoes not bind to EWS-FLI1 was expressed, no increase in colony formationwas seen (FIG. 3, yellow bar). In order to determine if this lack ofcolony growth was only due to the inability to bind to EWS-FLI1 orsecondary to RHA structural changes, ATPase activity was measured fromthe FLAG immunoprecipitated protein. The D827A mutation did not affectRHA function (FIG. 3B).

Peptide Binds to EWS-FLI1 and Blocks RHA from Binding to EWS-FLI1

An assay that will identify whether a peptide can disrupt RHA frominteracting with EWS-FLI1 was developed. The Biacore device is abiosensor that utilizes surface plasmon resonance (SPR) to measure thestrength of molecular interactions. SPR is the sensitive measurement ofdiffracted light based upon alterations of surface characteristics thatchange with molecules binding to a surface. The Biacore device combinesSPR with a microfluidic system. The y-axis demonstrates resonance units(RU), which is the position of a reflected beam of light at any giventime. The x-axis shows time in seconds.

Surface plasmon resonance (SPR) was used to demonstrate an interactionbetween EWS-FLI1 and a peptide containing the 10 amino-acids of RHA(E9R; SEQ ID NO: 29), identified from the phage experiment fused to thecell-penetrating peptide antennapedia (SEQ ID NO: 34), Antp-PPPLDAVIEA(E9RP, FIG. 4). The K_(D) of E9RP binding to EWS-FLI1 was calculated tobe 4.0 μM (FIG. 4).

E9RP was evaluated for the ability to block RHA from binding to EWS-FLI1using both competitive ELISA assay and solution immunoprecipitationassays. In addition, specificity of interaction was evaluated bydisrupting EWS-FLI1 binding from RHA without interfering with other RHAbinding proteins. Creb-binding protein (CBP, p300) is critical tocellular basal transcription, so it was determined if a peptide thatwould disrupt EWS-FLI1 binding from RHA could also disrupt CBP binding.

The EWS-FLI1 binding peptide, E9RP (SEQ ID NO: 29), was able to preventthe binding of RHA to EWS-FLI1 in a dose-dependent manner in this ELISAassay, with an IC50 of approximately 0.2 μM (FIG. 5, Panel A), whilelacking a dose-dependent effect upon the binding of CBP (aa 1680-1890)(FIG. 5, Panel B).

The data suggest that E9RP can prevent the interaction of EWS-FLI1 andRHA, both by ELISA (FIG. 5) and in solution by showing that theircomplex is disrupted by the peptide, E9RP (FIG. 6). The IC₅₀ to preventcomplex formation is approximately 0.1 μM, very similar to results fromthe ELISA assay (FIG. 5).

Single Amino Acid Substitutions in GST-RHA (630-1020) Reduce EWS-FLI1Binding.

Amino-acids in positions 824 and 827 appear to be the important forEWS-FLI1 binding. The region of RHA identified by the E9RP peptide wasmutated in the GST-RHA (630-1020) fusion. Five amino acids wereindependently mutated to alanine for immunoprecipitation studies (FIG.7A). Wild-type GST RHA (630-1020) showed a 2-fold increase overbackground, whereas P824A and D827A did not show increased binding overbackground (FIG. 7B). Thus amino-acids in positions 824 and 827 appearto be important for EWS-FLI1 binding, as their mutation results in areduction of complex formation (FIG. 7B). These mutations have allowedthe creation of a definitive RHA mutant that does not bind to EWS-FLI1as well as prepare control peptides for cell growth experiments.

RHA Peptide Inhibits Monolayer ESFT Cell Growth but not NeuroblastomaGrowth

Disrupting the RHA:EWS-FLI1 complex is believed to inhibit growth in anESFT cell line specific fashion. For these experiments, the RHA peptideidentified from the original phage display experiments was synthesizedwith an N-terminal tag containing the cell-permeable peptide (CPP)Penetratin (a.k.a. Antennapedia), a sequence used for transport intocells as well as N-ter fluorescein (E9RP) (Terrone D, Sang S L, RoudaiaL, Silvius J R. Penetratin and related cell-penetrating cationicpeptides can translocate across lipid bilayers in the presence of atransbilayer potential. Biochemistry 2003; 42(47):13787-99; Lindgren M,Gallet X, Soomets U, et al. Translocation properties of novel cellpenetrating transportan and penetratin analogues. Bioconjug Chem 2000;11(5):619-26; and Thoren P E, Persson D, Karlsson M, Norden B. Theantennapedia peptide penetratin translocates across lipid bilayers—thefirst direct observation. FEBS Lett 2000; 482(3):265-8). The 16 aaPenetratin peptide was derived from the Drosophila Antennapediahomeodomain that enhances uptake of peptides by cells (Derossi D, JoliotA H, Chassaing G, Prochiantz A. The third helix of the Antennapediahomeodomain translocates through biological membranes. J Biol Chem 1994;269(14):10444-50, Perez F, Joliot A, Bloch-Gallego E, Zahraoui A,Triller A, Prochiantz A. Antennapedia homeobox as a signal for thecellular internalization and nuclear addressing of a small exogenouspeptide. J Cell Sci 1992; 102 (Pt 4):717-22). Confocal imagingdemonstrated the E9RP peptide entering nearly 100% of the cells as wellas entering the nucleus of ESFT cell lines 1 and 2 as well as controlneuroblastoma cells (FIGS. 8A and 8B).

E9RP (10 μM) inhibited the growth of two ESFT cell lines 1 and 2, butdid not affect the growth of a neuroblastoma cell line that lacksEWS-FLI1 but expresses wild-type EWS (FIG. 8C). Two additional sarcomacell lines, lacking EWS-FLI1, were not growth inhibited by E9RP, yetboth exhibited significant nuclear uptake of the peptide (data notshown). Control peptides with the single amino acid substitutions shownabove demonstrated no effect on cell growth (FIGS. 8D, 8E). Theseresults support the concept of the specific inhibition of ESFT cellgrowth using a peptide directed at EWS-FLI1.

RHA Peptide Expressed in ESFT Cells Reduces Soft-Agar Colonies, but doesnot Reduce Rhabdomyosarcoma Colony Growth

Since the Antp peptides added to media would not remain in the cells for2 weeks during soft-agar colony growth, expression peptides were used toprovide continuous exposure to peptide. A pair of expression plasmidswith cloning sites downstream of the enhanced green fluorescent protein(EGFP) was obtained; the pG plasmid contained no localizing sequence andwas expressed throughout the cells (FIG. 9, left panels). The pGCplasmids contained a nuclear export sequence that blocked peptidetransport into the nucleus (FIG. 9, right panels). The E9R DNA sequencewas synthesized and the hybridized oligodeoxynucleotides were insertedinto the cloning site downstream of either EGFP or EGFP and nuclearexport sequence.

When E9R was expressed throughout ESFT (TC71) cells, soft agar colonygrowth was suppressed (FIG. 9, left upper). However, rhabdomyosarcomacells were not reduced by the E9R expression (FIG. 9, left lower). Whenthe E9R was excluded from the nucleus (pGCE9R), no growth reduction wasseen for either cell line (FIG. 9, right panels). Expressed E9R also didnot affect soft agar growth of neuroblastoma cells, also lackingEWS-FLI1 (not shown). This supports our conclusion about the specificeffects of E9R inhibition of ESFT cells and suggests that nuclearpeptide is required for an effect. Unfortunately, the empty vectorplasmid with the nuclear import signal was toxic to all cells,potentially from high levels of EGFP in the nucleus (not shown).

E9R Peptide Expression Reduces ESFT Murine Xenograft Growth

The ability of the E9R peptide sequence to prevent ESFT tumorigenesis ina pilot murine xenograft experiment was determined. As a controlpeptide, the same mutation that prevented wild-type RHA from binding toEWS-FLI1 (D827A, noted in the 10-mer peptide as E9R-D5A) was created.ESFT cells were transfected with either pGE9R or pGE9R-D5A byelectroporation. A transfection efficiency of greater than 80% usingfluorescent microscopy (FIG. 10A) was observed. Transfected cells,500,000 cells per mouse, were implanted into the gastrocnemius muscle of6 mice per group by syringe injection. Animals were observed for tumorgrowth, and were measured as soon as palpable tumors appeared after 14days. Every two to three days, each animal's thigh was measured in threedimensions, and tumor volumes were calculated. The tumor take-rate andrate of growth was greater in the animals whose tumors contained themutated peptide (E9R-D5A) (FIG. 10B). After 26 days, the animals withthe largest tumors began to limp and the experiment was ended (FIG.10C). Using a 2-way ANOVA analysis, followed by Bonferroni correctionfor multiple variables, the difference in the two growth curves washighly significant, p=0.016.

Small Molecule Screening Identifies a Lead Compound with High Affinityfor EWS-FLI1

Using the validated interaction between EWS-FLI1 and RHA as atherapeutic target, one logical progression is to identify smallmolecules in silico that could be predicted to prevent the interaction.Unfortunately, since EWS-FLI1 is a disordered protein (Ng K P, PotikyanG, Savene R O, Denny C T, Uversky V N, Lee K A. Multiple aromatic sidechains within a disordered structure are critical for transcription andtransforming activity of EWS family oncoproteins. Proc Natl Acad Sci USA2007; 104(2):479-84) and significantly hydrophobic (Uren A,Tcherkasskaya O, Toretsky J A. Recombinant EWS-FLI1 oncoproteinactivates transcription. Biochemistry 2004; 43(42):13579-89),3-dimensional structural information of the chimera is not available. Anapproach was chosen to screen small molecule libraries for compoundsthat bind to EWS-FLU. Recombinant EWS-FLI1 was bound to a CM5 sensorchip. 1000 compounds from the NIH, NCI, DTP library of compounds werescreened using a single concentration injection of each compound. Anexample of the screening from a portion of one plate containing 80compounds is shown (squares, FIG. 11). The data shown include theposition on a 96-well plate, the NSC compound number, the molecularweight and Rm normalization. An initial selection of promising compoundswas based on the Rm normalization. The Rm normalization is a ratio ofthe actual resonance unit binding measurement divided by the theoreticalmaximal binding. Ratios that exceed 2.0 likely indicate issues ofcompound solubility or polymerization. Ratios less than 0.7 suggest poorbinding with affinity that would not be adequate for blocking aprotein-protein interaction. The compounds that were selected aspotentially binding to EWS-FLI1 were further evaluated over a range ofconcentrations in order to identify a binding affinity (not shown).

Following Biacore T100 screening of 1000 compounds, one of the compoundswith outstanding binding kinetics (FIG. 12), both qualitatively andquantitatively demonstrated a unique 3-dimensional structure (FIG. 15).In a comparative modeling experiment, this compound, NSC635437, hadsurprisingly similar structure to our well-studied peptide sequence E9R(SEQ ID NO: 29). NSC635437 also has significant structural homology tothe first three prolines in our peptide using a ligand overlap strategy(FIG. 15). The combination of a significant binding affinity and asurprising structure will lead us to pursue a series of structuralmodifications to optimize binding and functional inhibition in futureproposals.

The small molecule lead compound was tested for the ability to block RHAfrom binding to EWS-FLI1 using a solution immunoprecipitation assay.NSC635437 not only has good binding to EWS-FLI1, but can block thebinding of RHA to EWS-FLI1 in solution binding assay at 10 μM (FIG.13A), consistent with the SPR measured binding affinity (FIG. 12). Inaddition, the lead compound was specifically cytotoxic to ESFT cells inculture compared with neuroblastoma cells (FIG. 13B).

Compound NSC635437 has Homology to Peptide E9R

A 3-dimensional model of the E9R peptide was developed to determine ifany of our lead compounds might have structural similarity. A ligandoverlap strategy was used by first sampling the Brookhaven database forx-ray structures that contained the PPPLDAVIEA (SEQ ID NO; 29) motif andBLAST alignment was made for the 10-mer PPPLDAVIEA (SEQ ID NO; 29)sequence to predict the structure. The best sequence alignment (FIG. 14)was extracted and predicted the structure using homology modeling (1)with a SQUALENE-HOPENE-CYCLASE (PDB: 1SQC) x-ray structure as atemplate. The input alignment for the Modeler (1) was obtained withBLAST (2).

Subsequently, to confirm the correct orientation of the mutatedresidues, BLAST was counter searched for the sequence motif ‘PPPLD’ (SEQID NO: 32). Several hits were obtained and five structures weresuperimposed together with the 10-mer peptide (FIG. 15). The predictedorientation matches well with the x-ray orientation obtained for the‘PPPLD’ sequence (SEQ ID NO: 32) with a minimum root mean squaredeviation (RMSD) of 0.62 A and maximum RMSD of 1.69 Å. Using a multi-fitprocedure, a fit of the atom coordinates of the lead NSC635437 onto atomcoordinates of the inhibitory peptide motif (FIG. 15) was searched for.NSC635437 (identified from the Biacore screening, FIG. 11 to have aK_(D) of 2.34 μM) provided an excellent mimic of the PPP portion of theE9R motif (see FIG. 15B). Based on this fit, decoration of NH of thelactam ring of NCS635437 with peptide motifs to mimic the LDAVIEA (SEQID NO: 33) portion of PPPLDAVIEA (SEQ ID NO; 29) can be performed, andpharmacophores within PPPLDAVIEA (SEQ ID NO; 29) defined byphysico-chemical descriptors (i.e., distance, H-bond donor acceptors)can be generated. A preliminary 3-D pharmacophore model (FIG. 15C) wasgenerated using the UNITY module of Sybyl 7.0 (Tripos Inc., St. Louis,USA) by assigning hydrogen bond donors and acceptors. Screening of avirtual database of 55 million compounds has begun, and the basic coreof NSC635437 (FIG. 16) has been synthesized.

Results

Data show that RHA binds directly to EWS-FLI1 (FIGS. 1 and 2), and thatthis interaction leads to functional modulation (FIG. 3). Based uponthis discovery, the E9R peptide is a useful tool to probe the binding ofEWS-FLI1 to RHA. The E9RP binds to EWS-FLI1 (FIG. 4) and can disrupt RHAfrom binding to EWS-FLI1 (FIGS. 5 and 6). The growth of ESFT cells, butnot other embryologic tumors, is reduced by the E9RP (FIG. 8) and theexpressed E9R reduces soft-agar colony and xenograft tumor formation inESFT but not other tumors (FIGS. 9 and 10).

Enhanced binding studies have been demonstrated with small moleculesusing a Biacore T100 (FIGS. 11, 12, 23, 24). This identified smallmolecule lead compound, identified from SPR analysis of a library ofcompounds is proof-of-principle of the general approach (FIGS. 13-15).The effect of peptide E9R blocking RHA from binding to EWS-FLI1 isestablished as a comparative signature and ‘gold-standard’ for smallmolecules. A lead compound has been developed and a series of activesmall molecules that specifically disrupt EWS-FLI1 regulatedtranscription have been discovered.

Additional Experiments Involving the Peptide

E9R can be used as a tool to disrupt EWS-FLI1 from interacting with RHAand measure the consequences of that disruption. The effects ofdisrupting EWS-FLI1 from RHA on the full ESFT transcriptome can beevaluated. A cDNA signature of the E9R peptide treated ESFT cells can beestablished and compared to the small molecule lead compounds.

The growth effects of the E9R peptide upon ESFT cells can be determinedin comparison with a panel of non-transformed cell lines. The peptidecan be tested in animal models of ESFT to validate whether disruption ofEWS-FLI1 from RHA will result in decreased ESFT tumor growth, reductionof established tumors, and the cellular biochemical effects of thisdisruption. NSC635437 can be tested to compare the effectiveness of thesmall molecule with the peptide ‘gold standard’.

Modifications of NSC635437 can be made and the resulting compoundstested using surface plasmon resonance for EWS-FLI1 binding kinetics andin solution RHA binding experiments. Compounds that have improvedparameters compared to the lead compound can be tested in cDNA array,cell based assays, and animal tumorigenesis.

EGFP-peptide expression vectors can be transfected into ESFT A673 cells.To directly examine genes regulated by EWS-FLI1, the first step is togrow a population of EWS-FLI1 bearing A673 Ewing's sarcoma cells. Theexperiment can be done with A673 cells. The signature for these cellsboth expressing EWS-FLI1 and an inducible siRNA that eliminates EWS-FLI1expression have been validated in multiple U133 Affymetrixoligonucleotide arrays. After sufficient numbers are grown, this cellline can then be transfected with EWS-FLI1 interacting peptideconstructs (FIG. 9) as well as the mutant controls.

A population of EGFP positive cells can be selected and RNA harvested.After a recovery period following transfection, flow sorting can be usedto select for successfully transfected cells. These cells can be grownin a “carry plate.” When a sufficient number of cells has been grown,the flow sorted cells can then be plated out at ˜9×10⁶ cells per 15 cmplate. RNA can then be harvested approximately one day after uniformplating. This can be repeated a total of three times for eachtransfected line, resulting in nine total cell pellets. Harvested cellpellets can be processed with RNeasy Mini Kit (Qiagen). RNA can then bequantified and frozen for transport and further processing.

Once received, the RNA samples can be analyzed for quality via theBioAnalyzer, and then processed with the Affymetrix probe preparationkits. These samples can then be hybridized to Affymetrix U133PlusGeneChips. “Signatures” from peptide-treated cells can be obtained usingthe an approach to derive the EWS-FLI1 signatures (Smith R, Owen L A,Trem D J, et al. Expression profiling of EWS/FLI identifies NKX2.2 as acritical target gene in Ewing's sarcoma. Cancer Cell 2006; 9(5):405-16).Thus, genes can be sorted using the signal-to-noise metric followed bypermutation testing. Both up- and down-regulated gene sets can beidentified in this manner. Comparisons between the peptide-inducedsignatures and the EWS-FLI1 transcriptional profile can be performedusing two complementary approaches. In the simplest approach, Chi squareanalysis can be used to determine whether the overlaps in each gene setare greater than expected by chance alone. In the second approach, GeneSet Enrichment Analysis (GSEA; (Smith R, Owen L A, Trem D J, et al.Expression profiling of EWS/FLI identifies NKX2.2 as a critical targetgene in Ewing's sarcoma. Cancer Cell 2006; 9(5):405-16)) can be used tocompare datasets. GSEA measures the enrichment of one gene set near thetop of a second rank-ordered gene list, and quantifies this enrichmentusing a running sum statistic called the enrichment score (Smith R, OwenL A, Trem D J, et al. Expression profiling of EWS/FLI identifies NKX2.2as a critical target gene in Ewing's sarcoma. Cancer Cell 2006;9(5):405-16). The EWS-FLI1-regulated gene list will be order ranked, andwhether the peptide-derived signature is enriched near the top of thisrank-ordered list will be determined. This second approach is useful asit tends to identify correlations that are more subtle, or difficult toidentify due to noise in the system (Smith R, Owen L A, Trem D J, et al.Expression profiling of EWS/FLI identifies NKX2.2 as a critical targetgene in Ewing's sarcoma. Cancer Cell 2006; 9(5):405-16). The Lessnicklab has used both of these approaches to compare distinct microarrayexperiments (Lessnick S L, Dacwag C S, Golub T R. The Ewing's sarcomaoncoprotein EWS/FLI induces a p53-dependent growth arrest in primaryhuman fibroblasts. Cancer Cell 2002; 1(4):393-401; Smith R, Owen L A,Trem D J, et al. Expression profiling of EWS/FLI identifies NKX2.2 as acritical target gene in Ewing's sarcoma. Cancer Cell 2006; 9(5):405-16;Kinsey M, Smith R, Lessnick S L. NROB1 is required for the oncogenicphenotype mediated by EWS/FLI in Ewing's sarcoma. Mol Cancer Res 2006;4(11):851-9). Furthermore, by obtaining the “leading edge” gene listsfrom the GSEA analysis (that is, the genes that are most correlatedbetween the two experiments; (Subramanian A, Tamayo P, Mootha V K, etal. Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles. Proceedings of theNational Academy of Sciences of the United States of America 2005;102(43):15545-50)), genes can be identified that are most likely to beregulated by the RHA-EWS-FLI1 interaction. The net result is that it canbe determined how much, and which portion, of both the EWS-FLI1 and RHAsignatures are due to the interaction between the two proteins.

Signatures can be used for small molecule development. Once thesignature of peptide E9R that prevents RHA from binding to EWS-FLI1 isidentified, the small molecules can be tested to see if they havesimilar signatures to peptide E9R and siRNA reduction of EWS-FLI1. Thiscomparison can be used as additional lead compounds are identified, andthose molecules that most closely resemble the EWS-FLI1 reduction orpeptide treated signatures can be considered as mechanistic probes. ThecDNA comparison signatures can be used in the prioritization ofcompounds. For example if a derivative of NSC635437 or a new compoundhas a signature that is markedly different from the E9R peptide, it cansuggest a broader, and likely, less specific mechanism. If a signatureis qualitatively different, it will suggest alternate mechanisms. Thisdata can be used in both compound modification and later for toxicityevaluation.

The E9R peptide can be validated for specificity in additional ESFT andnon-ESFT cell lines. E9R can be synthesized with the Antennapediasequence (E9RP). These peptides can be tested against a panel of ESFTcell lines (TC32, TC71, SK-ES1, A4573, RD -ES, and ES0925) for growthinhibition and soft-agar colony formation as previously published (AbaanO D, Levenson A, Khan 0, Furth P A, Uren A, Toretsky J A. PTPL1 is adirect transcriptional target of EWS-FLI1 and modulates Ewing's Sarcomatumorigenesis. Oncogene 2005; 24(16):2715-22). Testing can also occur incancer cell lines that lack EWS-FLI1 including neuroblastoma andleiomyosarcoma. NON-TRANSFORMED control cell lines will be used fortesting specificity including primary fibroblasts, MCF-10A epithelialcells, and HEK293 kidney cells. Since the ESFT cell of origin issuggested as a mesenchymal stem cell (MSC), these MSC can be preparedfrom murine bone marrow as a final control of normal cells(Castillero-Trejo Y, Eliazer S, Xiang L, Richardson J A, Ilaria R L, Jr.Expression of the EWS/FLI-1 oncogene in murine primary bone-derivedcells Results in EWS/FLI-1-dependent, Ewing sarcoma-like tumors. CancerRes 2005; 65(19):8698-705; Riggi N, Suva M L, Stamenkovic I. Ewing'sSarcoma-Like Tumors Originate from EWS-FLI-1-Expressing MesenchymalProgenitor Cells. Cancer Res 2006; 66(19):9786). Given the challenge ofpreparing these MSC, they are only used for specificity studies of themost promising compounds. Control peptides can be those with singleamino acid substitutions. Compounds can be tested for effects instandard 96-well growth assays (FIG. 8) and for their ability toinitiate apoptosis using caspase-3 cleavage, as previously published(Toretsky J A, Thakar M, Eskenazi A E, Frantz C N. Phosphoinositide3-hydroxide kinase blockade enhances apoptosis in the Ewing's sarcomafamily of tumors. Cancer Res 1999; 59(22):5745-50).

The in vivo anti-tumor effect of peptides and small molecules thatprevent EWS-FLI1 and RHA interaction can be tested. It is believed thatdemonstrating efficacy of hitting the molecular target can lead toreduced tumor growth in an animal model. Data was obtained with celllines constitutively expressing the peptides. While this is useful asproof-of-concept, these derived cell lines may ultimately undergomutation to allow their optimal growth, and therefore, native ESFT cellsthat are grown as xenografts can be useful. Xenografts can then betreated with injected peptides. There is a significant amount ofliterature suggesting that modified peptides can be injectedintraperitoneally or intravenously in mice with resultant uptake intumors (Toretsky J A, Thakar M, Eskenazi A E, Frantz C N.Phosphoinositide 3-hydroxide kinase blockade enhances apoptosis in theEwing's sarcoma family of tumors. Cancer Res 1999; 59(22):5745-50;Walensky L D, Kung A L, Escher I, et al. Activation of apoptosis in vivoby a hydrocarbon-stapled BH3 helix. Science 2004; 305(5689):1466-70).This aim will test the optimized peptides for their distribution inmurine tissue, ability to disrupt EWS-FLI1 from RHA in growing tumors,and ability to prevent or reduce tumor growth.

Tissue distribution and dose finding experiments can be conducted.SCID/bg can be injected with fluorescein and Antennapedia labeledpeptides. A dose escalation of 3, 10, 30, and 100 mg/kg based uponsimilar peptide dosing in the literature (Walensky L D, Kung A L, EscherI, et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3helix. Science 2004; 305(5689):1466-70) can be employed. Six mice can beinjected by tail vein injection at each dose. Two mice from each groupcan be euthanized at 8 and 24 hours and can undergo full necropsy toidentify the distribution of peptide. Tissue sections from major organscan be analyzed for distribution of fluorescent peptide. If significantpeptide is found at 24 hours, then a follow-up experiment can use thehighest concentration of peptide that achieves good tissue levelswithout toxicity to the mouse (loss of 10% body weight or neurologicdysfunction). In this follow-up experiment, six mice per time point candetermine the kinetics of tissue distribution. Time points can include24, 48, and 72 hours. Dose finding experiments can require 24 mice andtime course experiments can require 18 mice. Tissues can be analyzed fortoxic effects of peptide using standard histological analysis. Thisexperiment can be repeated once, for a total of 84 mice.

Pharmacodynamic effect of E9RP on in vivo disruption of EWS-FLI1 and RHAcan be investigated using a model of ESFT in a nude mouse that consistsof orthotopic placement (gastrocnemius) of tumor, as described in detailbelow (Merchant M S, Woo C W, Mackall C L, Thiele C J. Potential use ofimatinib in Ewing's Sarcoma: evidence for in vitro and in vivo activity.J Natl Cancer Inst 2002; 94(22):1673-9). Seven days following tumor cellinjection, animals can be treated with daily i.v. injections of E9R P.Eight hours following the third injection, a subgroup of animals can beeuthanized and undergo necropsy. Eight hours following an injection canbe used to maximize the potential of high peptide concentration anddissociated EWS-FLI1 and RHA. Gross specimen tumors can be measured forsize and mass; however, the primary endpoint is biochemical dissociationof EWS-FLI1 and RHA. Tumors can then be split, with approximately ⅔snap-frozen in liquid nitrogen and ⅓ preserved in formalin for paraffinembedding. Other organs with fluorescent peptide distribution, asdetermined above, can be preserved for toxicologic and immunologicevaluation.

Tumors can be analyzed for signature effects of peptide. Paraffinembedded tissue can undergo sectioning followed by routine hematoxylinand eosin staining as well as TUNEL (for apoptosis), andimmunohistochemistry for expression of EWS-FLI1, RHA, PTPL1, TGFβRII,and markers of ESFT such as CD99. Additional immunohistochemistry canevaluate tumor and organ vascularization, techniques recently published(Torchia E C, Boyd K, Rehg J E, Qu C, Baker S J. EWS/FLI-1 Induces RapidOnset of Myeloid/Erythroid Leukemia in Mice. Mol Cell Biol 2007;27(22):7918-34). These studies can allow determination of whether theprotein levels of EWS-FLI1 targets are affected by the peptides. Theapoptosis index can provide information about the tumor toxicity of thepeptides and potential toxicity to non-tumor tissues. Snap-frozen tissuecan be pulverized under liquid nitrogen and divided for extraction ofRNA or protein, as previously published (Toretsky J A, Zitomersky N L,Eskenazi A E, et al. Glypican-3 expression in Wilms tumor andhepatoblastoma. J Pediatr Hematol Oncol 2001; 23(8):496-9). RNA can beevaluated by quantitative RT-PCR for expression of ESFT target mRNA suchas ID2, PTPL1, TGFβRII, and p21^(Waf/CIP). Protein can beimmunoprecipitated with EWS and FLI1 antibodies as demonstrated (FIG. 6)to determine if the peptide has prevented the association of EWS-FLI1from RHA in vivo.

Primary tumor growth effects can be investigated using a model of ESFTin a SCID/bg mouse that consists of orthotopic placement of tumor asdescribed in detail below and has been recently published (Merchant M S,Yang X, Melchionda F, et al. Interferon gamma enhances the effectivenessof tumor necrosis factor-related apoptosis-inducing ligand receptoragonists in a xenograft model of Ewing's sarcoma. Cancer Res 2004;64(22):8349-56). A luciferase gene can be transfected under aconstitutive promoter into an ES0925 ESFT cell line. Peptides can beadministered as determined above every other day. The top two peptidesand a control based on their in vitro activity can be administered sevendays after injection of the tumor cells. ESFT injected mice developtumors with approximately 90% efficiency following tumor injection (datanot shown). The diameter of the tumors can be measured at 3 dayintervals, beginning at on day 5 post-inoculation with 1×10⁶ Ewing'sSarcoma tumor cells injected into the gastrocnemeous. After beginningtreatment the tumor size can be measured daily until the completion ofthe study. Intraperitoneal luciferase can be injected followed byXenogen imaging every 4-5 days to both monitor primary tumor volume andassess for metastatic lesions. Mice can be treated until the tumorreaches a volume of 1.0 cm³. The majority of untreated mice can beexpected to reach this volume in two to three weeks. The tumor can beresected during a survival surgery that has been IACUC approved andpreviously published (Merchant M S, Woo C W, Mackall C L, Thiele C J.Potential use of imatinib in Ewing's Sarcoma: evidence for in vitro andin vivo activity. J Natl Cancer Inst 2002; 94(22):1673-9). The size ofthe tumor can be measured and weighed upon removal and then subdividedfor preservation as both snap frozen tissue and formalin-fixed tissue.Histopathologic evaluation of hematoxylin and eosin stained sections canbe conducted on the tumor, or on serial sections at the site ofinoculation, as appropriate based on observations at the grossdissection examination. The tumor can be examined for histologicalfeatures of neoplasia and pleomorphism, invasion and evidence ofapoptosis.

Sample size for localized tumor growth effects of peptide can beinvestigated. Mice can be randomized into two groups 7 days after theinjection, and receive either a control or an active peptide. Mice canbe observed 3 times per week, and the tumor incidence between the twogroups can be compared using a Chi-square test or Fisher's exact test.The tumor incidence of the control and the active-peptide groups areexpected to be 90-100% and 30%, respectively. Table 3 gives the numberof samples per group needed to detect a statistically significantdifference in tumor incidence between the two groups at the 5% level.For example, if one assumes that the tumor incidence of the two groupsare 95% and 30% respectively, 8 mice per group will provide 86% power todetect a statistically significant difference in tumor incidencesbetween the two groups at the 5% level. A first experiment can use 10mice per group, with adjustment in the follow-up experiment based uponresults.

TABLE 3 Incidence for controls (%) 90 90 90 90 95 Incidence foractive-peptide 50 40 30 30 30 group (%) Power (%) 79 82 81 85 86 N pergroup 18 13 9 10 8

Anti-metastatic activity of the peptide can be investigated. Followingresection of primary tumors as detailed above, mice are rendered free ofgross disease. Within 6-9 weeks, greater than 90% of untreated mice candevelop metastastic Ewing's tumors in the lungs, bones, subcutaneoustissues, and abdomen. These tumors are first detectable on clinical examof the mice with palpation and assessment of inducible dyspnea. Mice canbe assessed a minimum of 3 times per week after resection of the primarytumor for signs and symptoms of recurrent disease. Mice determined to behindered by metastatic disease can be sacrificed and tissues examinedfor histologic evidence of Ewing's sarcoma. As above with primarytumors, gross tumor can be snap frozen and formalin fixed for furtheranalysis of EWS-FLI1 expression. Mice can be followed for metastaticdisease following treatment during primary tumor growth. In addition, asecond grouping of mice can receive no treatment during primary growth,but can be treated with 3 weeks of peptide administration starting 7days after primary resection. This treatment during a stage of minimalresidual disease can mimic the opportunity in the clinical to treat highrisk patients following standard of care therapies. Ten mice per groupcan be followed for subsequent development of metastatic disease.Determination of power is equivalent to the calculations for primarydisease as above. Intraperitoneal luciferase can be injected followed byXenogen imaging every 4-5 days to both monitor metastatic lesions.

The ability of peptide to shrink existing tumors can be investigated.SCID/bg mice can be injected orthotopically with ESFT tumor cells. Whenthe tumors reach 0.125 cm³, the mice can be randomized into thefollowing two treatment groups: control peptide, active peptide. Themice can be observed 3 times per week, and tumor volumes 1 week afterthe treatment are expected to be 0.5 cm³ and 0.25 cm³ for the controland treated groups, respectively. It can be assumed that the two groupshave equal standard deviations. Table 4 gives the number of mice pergroup needed to detect a statistically significant difference in tumorvolumes at 1 week after the treatment at the 5% level. Tumor growthrates can be estimated by a linear mixed regression model with repeatedmeasures for each mouse, and the difference in slopes can be testedusing the p-value for the coefficient associated with the treatmentgroup. Appropriate transformations can be applied (log orarcsine-square-root) as needed to ensure normality. Sample size can beselected with Ying Zhang (statistician) after the above experiments.Animals can be euthanized and necropsy procedure as described above.

TABLE 4 Mean volume for controls (cm³) 0.5 0.5 0.5 Mean volume fortreatment groups (cm³) 0.25 0.25 0.25 Standard deviation for both groups0.15 0.15 0.2 Power (%) 82 91 75 N per group 7 9 10

Additional Experiments Involving Small Molecules

A small molecule has been identified by screening the NCI DTP library ofcompounds for molecules that bind to EWS-FLIT. The small molecule hassignificant 3-dimensional homology to the first 3 amino acids of thefunctional peptide, E9R. This small molecule has been used to design fortesting a series of small molecules for the ability to bind to EWS-FLI1and prevent or displace RHA. Derivatives can be evaluated foreffectiveness in cDNA array, cell growth and motility, and tumorigenesisassays.

Derivatives can be synthesized as outlined herein (FIG. 18). Ascompounds are prepared, they can be evaluated for EWS-FLI1 binding andthe ability to prevent RHA binding. Based upon these binding studies,chemical modifications can be designed and synthesized. As the compoundsdevelop stronger affinities for EWS-FLI1 and prevent RHA binding atlower concentrations, compounds can be advanced to cell based assays.Those compounds with reasonable drug-like properties (i.e. soluble, logP<5, structurally stable) and an IC₅₀ less than 2 μM can be evaluatedagainst ESFT cell lines. Compounds that demonstrate an enhanced activityprofile against transformed cells as compared with non-transformed cellscan be advanced to xenograft studies.

Superimposing structures between NSC635437 and peptide E9R (FIG. 15C)have been identified. In the strategy depicted in FIG. 17, fourmedicinal chemistry directions are implemented to find inhibitors basedon the inhibitory peptide sequence PPPLDAVIEA and to develop biochemicaltools to study EWS-FLI1 interactions. Eight peptides fragments (9-mersto 2-mers) are synthesized to investigate the importance of the P and Damino acid arrangement in the active site. Overlap can be modeledbetween peptides and synthesized compounds. This overlap strategy canallow prediction of enantiomers that might lack effectiveness, testthose compounds, and further support our modeling. Non-effectiveenantiomers can act as outstanding control small molecules for cellulartoxicity studies. The preparation of fluorescent peptide ligands can beuseful for in vitro studies that can measure displacement upon smallmolecule binding. This can provide alternative and potentially effectivemeans of screening for small molecule binding to EWS-FLI1 by causingpeptide displacement. This can allow confirmation that the actuallocation of small molecule binding was the site of peptide binding aswell as cellular localization and peptide penetration into the cells.

A lead compound that binds to EWS-FLI1 with a K_(D) of 2 μM has beenidentified. Based on the lead NCS635437, seven series of analogues havebeen designed in order to optimize this scaffold (FIG. 18). Series A, Band E represents optimization of the aromatic rings. Series C attemptsto elongate the lead NCS635437 to approximate the LDAVIEA portion of thepeptide (see FIG. 15C for overlap). An example of the conjugated ligandis shown in FIG. 21. Series D represents enantiomers of NCS635437.Series F provides for a dansylated derivative of the lead for use as abiochemical tool. These dansylated small molecules can be used toevaluate compound uptake, intracellular kinetics, and localization incells. Finally, series G represents a dehydrated NCS635437 to evaluatethe importance of the hydrogen bonding component.

The synthesis of the basic core of NSC635437 is shown in FIG. 16. Thesynthetic strategy can be easily adapted using known synthesistechniques to functionalize all key analogues proposed (FIG. 18), aswill be appreciated by one of skill in the art.

Because NSC635437 is a chiral compound, a synthetic strategy to makeeach enantiomer (FIG. 21) has been developed. High enantioselectivitywas achieved by addition of diethylzinc to N-methylisatin in thepresence of the DBNE catalyst (FIG. 20). A high resolution x-ray crystalstructure is provided assigning the R-configuration to the (−)enantiomer of 31 (FIG. 20). Recently, Ojida (Ojida A, Yamano T, Taya N,Tasaka A. Highly enantioselective reformatsky reaction of ketones:chelation-assisted enantioface discrimination. Org Lett 2002;4(18):3051-4) and co-workers showed that a high enantioselectiveReformatsky reaction with ketones could be achieved using cinchonine asa chiral additive. As shown in FIG. 21, addition of the Reformaskyreagent 32 in the presence of 1.5 equivalents of cinchonine and 4.0equivalents of pyridine to aromatic ketone 32, gave the tertiary alcohol33 in 97% yield and 97% ee.

Enantioselective organozinc addition to isatin (FIG. 20) has beensuccessfully performed, and results of enantioselective Reformatskyaddition into ketones (FIG. 21), has been published, indicating thatsynthesis of non-racemic isatin derivatives by enantioselectiveReformatsky-like reaction (FIG. 22) can be achieved. The Reformatskyreagent 34 derived from the aryl bromomethyl ketone in the presence ofcinchonine and pyridine is expected to give non-racemic isatinderivative 24 in high ee. Protection of 28 may be required and chemistrycan be adapted based on initial results.

Small molecules can be evaluated for the ability to bind to EWS-FLI1 andprevent its binding to RHA. The Biacore T100 can be used to establishthe binding kinetics of small molecules to EWS-FLI1 (FIGS. 4, 12, 23 and24). Each of the small molecules generated can undergo kineticevaluation for binding to EWS-FLI1. In addition, all synthesizedmolecules can be tested for the ability to disrupt EWS-FLI1 from RHAusing solution immunoprecipitation (FIGS. 6 and 13) and ELISA (FIGS. 1and 5). Control proteins can include other RHA binding proteins such asCBP (FIG. 5).

Additional iterative cycles of modeling, chemistry, bioassay, andrational modifications can be done to the lead structures in order tooptimize binding and develop molecules that reasonably fit criteria forpharmaceutical agents, namely, that they exhibit a drug-like profile andfulfill standard guidelines such as Lipinski's rule of 5. Compounds caninitially be prioritized based on the following sequence: (1) strengthof binding to EWS-FLI1 with dissociation of RHA, (2) specificity oftoxicity to ESFT cell lines, and (3) drug-like properties. When thecompounds bind EWS-FLI1 with low nanomolar affinity and exhibit specificcytotoxicity, then drug-like properties for optimized delivery andminimized toxicity will be pursued.

Small molecules can be tested in ESFT cell lines for toxicity inmonolayer and soft-agar growth. A panel of ESFT and non-transformed celllines is described herein that can be used to determine the cellulartoxicity of developed small molecules. These can be applied to advanceagents into small molecule animal testing to identify one or more newchemical entities capable of inhibiting ESFT oncogenesis in vivo whileexhibiting appropriate pharmacokinetic parameters and limited toxicityto normal cells. The toxicity to cells can be correlated with theability to disrupt EWS-FLI1 from RHA in order to advance to newiterations of small molecules. Control molecules can include inactiveenantiomers identified herein. Control cell lines can includenon-transformed epithelial and mesodermal cultures as well as non-ESFTtumor cell lines.

Small molecule testing in ESFT xenograft models can be conducted. Thesemolecules can be tested in animal studies. Dosage can be based upondetermining the MTD using toxicity criteria (10% weight loss orneurologic dysfunction) and then dose reductions from 67% of the MTD intumor xenograft studies to find the minimal useful dose. Serum, tumorand non-tumor tissues can be analyzed for compound levels at timepoints0,1,2,4,6,12, and 24 following a single injection of compound usingHPLC. Additional pharmacokinetic/pharmacodynamic studies can beperformed.

Recombinant EWS-FLI1, EWS, and FLI1 can be produced. RecombinantHis(6×)-EWS-FLI1 can be prepared using an expression system in BL21 E.coli and purification of His-tagged protein using a Ni-charged columnfollowed by an on-column re-folding protocol. This is both a time andlabor intensive component of purification. Since the protein is onlyuseful for two weeks, a constant supply is required and thus a reliableprotein purification apparatus is required. This strategy provides thebest quality, transcriptionally active EWS-FLI1 (Uren A, TcherkasskayaO, Toretsky J A. Recombinant EWS-FLI1 oncoprotein activatestranscription. Biochemistry 2004; 43(42):13579-89). Experience withrecombinant EWS-FLI1 indicates that protein is optimal for use for 3-4weeks. Similar vectors have been prepared and transformed into BL21cells with full length EWS and FLI1. Both recombinant proteins havesuccessfully been prepared.

Bioinformatics of small molecule array can be investigated. Each proteinis screened against three replicate microarrays and fluorescenceintensities are calculated for each printed feature. Composite Z-scoresare computed for the replicate datasets and used to rank compounds forfurther characterization. Promiscuous binders are filtered from the dataset and each compound is ranked for specificity of binding against apanel of nearly 250 proteins unrelated to EWS-FLI1. Compounds thatreproducibly bind to the protein in a specific manner are considered foranalysis in SPR assays.

Small molecule activity can be confirmed using a secondary screen forESFT cytotoxicity and measurements of surface plasmon resonance (SPR)assay for direct EWS-FLI1 binding. The secondary screen for smallmolecules that bind to EWS-FLI1 on the glass slide array can be ESFTcellular cytotoxicity assays. SPR allows for a tertiary screen toidentify the highest-affinity compounds for EWS-FLI1. It is expectedthat if 1% of screened small molecules show EWS-FLI1 binding, furtherevaluation of up to 1000 compounds will be required, which is feasiblein multiwell cytotoxicity assays. SPR can evaluate 1000 compounds fromthe NIH DTP library diversity and natural product sets. Thus, ascompounds are identified from the glass-slide array screen, they can beadvanced to cytotoxicity testing followed by SPR analysis. Thosecompounds that have relatively specific ESFT cytotoxicity and strongbinding kinetics to EWS-FLI1 (K_(D) of less than 10 microM) can beadvanced for further characterization. The additional evaluation ofcompounds that bind to EWS-FLI1 can include (i) EWS-FLI1 functionalinactivation and (ii) screening for the ability to inhibitprotein-protein interactions with RHA.

Assays for cell growth can be employed. Parallel plates of TC32 (ESFT)and NON-tumorigenic mouse embryonic fibroblasts (MEF), kidney (HEK293),and breast epithelial (MCF-10A) cells can be prepared with triplicateconcentrations of compounds. If four concentrations of compound (30, 3,0.3, 0.03 μM) are used, then six compounds are tested per plate perassay. In a semi-automated assay, if 16 plates (4 plates for each of 4cell lines) are set up per day, then 24 compounds could be tested perday (6 compounds per plate×4 plates per cell line=24 compounds per cellline). Cells are grown for 72 hours and viable cell number is estimatedfollowing incubation with MTT reagent. Solubilized dye will be measuredspectrophotometrically. Cell number is estimated from standard curvesestablished for each cell line. Some compounds may react chemically withthe dye and this would be apparent from the absorbance readings. Thosecompounds can be retested using alternate cell viability methods, suchas BrdU uptake.

A biochemical screen for target specificity can be conducted. Smallmolecules that demonstrate ESFT cytotoxicity and a K_(D) of binding toEWS-FLI1 in the nanomolar range can be tested for the ability to blockRHA from binding to EWS-FLI1. These experiments can be performed forboth peptide (FIG. 6) and small molecule (FIG. 13). The biochemistryexperiments can help to establish mechanism for novel compounds.

A general screen of EWS-FLI1 function can be performed. An ESFTcell-based assay for testing the transcriptional activity of EWS-FLI1oncoprotein can be developed. An EWS-FLI1 responsive promoter sequenceupstream of a short half-life green fluorescent protein coding sequenceand a nonspecific promoter that does not respond to EWS-FLI1 upstream ofa short half-life red fluorescent protein coding sequence cab be stablypresent in ESFT cells. Cells with both functioning promoters would beidentified as yellow in a fluorescent assay. This assay can be validatedwith the E9R peptide and siRNA against EWS-FLI1, such that successfulreduction of EWS-FLI1 function causes cells to turn red, indicating aspecific effect on EWS-FLI1 transcriptional activity but no effect onCMV viral promoter activity. Compounds with non-specific activitydiminish both red and green signals.

Example

Many sarcomas and leukemias carry non-random chromosomal translocationsencoding mutant fusion transcription factors that are essential to theirmolecular pathogenesis. These tumor-specific proteins can be employed inthe development of highly selective anticancer drugs. A particularlyclear example is provided by Ewing's Sarcoma Family Tumors (ESFT) whichcontain a characteristic t(11; 22) translocation leading to expressionof the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disorderedprotein that precluded standard structure-based small molecule inhibitordesign. Using surface Plasmon resonance screening, a lead compound,NSC635437, was identified.

YK-4-279, a para-methoxy derivative of NSC635437, blocks RHA binding toEWS-FLI1, induces apoptosis in ESFT cells, and reduces the growth ofESFT orthotopic xenografts. These findings demonstrate inhibition of theinteraction of mutant cancer-specific transcription factors with thenormal cellular binding partners required for their oncogenic activityand suitability for use as uniquely effective, tumor-specific anticanceragents.

Methods

E9R peptide was obtained from Bio-synthesis Inc, Lewisville, Tex.Protein G beads (Invitrogen, Carlsbad, Calif.), anti-GST, anti-FLI1,anti-Cyclin D1 antibodies (Santa Cruz, Calif.), Ac-DEVDAMC, caspase-3fluorogenic substrate (BD Biosciences Pharmingen), and anti-CleavedCaspase 3 (Asp175) (Cell Signaling) were commercially obtained.

Site Directed Mutagenesis

Every non-alanine amino acid in aa 823-832 region of RHA changed toalanine by site directed mutagenesis by using QuickChange II XLSite-Directed Mutagenesis Kit (Stratagene, Cedar Creek, Tex.) accordingto manufacturer's protocol.

Cell Cultures

Established TC32, TC71, A4573, CHP-100 and primary ES925 and GUES1 ESFTcell lines were maintained in RPMI (Invitrogen) media supplemented with10% FBS (Gemini Bioproducts). HEC and HFK cell lines, previouslydescribed (Uren, A., et al. Activation of the Canonical Wnt Pathwayduring Genital Keratinocyte Transformation: A Model for Cervical CancerProgression. Cancer Res 65, 6199-6206 (2005)). Stably EWS-FLI1expressing subclones of these cells were tested in anchorage independentgrowth assay as described previously (Toretsky, J. A., et al.Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res66, 5574-5581 (2006)).

Protein Immunoprecipitation Assays

Protein lysates and immunoprecipitations were performed as previouslypublished (Uren et al (2005)). Recombinant GST-RHA (647-1075) wasprepared from crude bacterial extracts without further purification.

Small Molecule Library Screening and Selection of Lead Compound

A surface plasmon resonance assay using the Biacore T100 was establishedwith EWS-FLI1, prepared in our laboratory as previously published (Uren,A., Tcherkasskaya, O. & Toretsky, J. A. Recombinant EWS-FLI1 oncoproteinactivates transcription. Biochemistry 43, 13579-13589 (2004)). DNAoligonucleotides were used to quality control the proper conformation ofEWS-FLI1 on the surface of a CM5 chip. Small molecules obtained from theDevelopmental Therapeutics Program of the National Cancer Institute, NIH(http://dtp.nci.nih.gov/branches/dscb/repo_open.html) were prioritizedbased upon their molecular weight and solubility. An initial screeningof molecules was performed at 1 or 10 μM compound, based on solubility.We used a model that compares the actual binding maximum (actual RU)with the theoretical binding maximum (RUtheor). If the RUactual toRUtheor is 0.9-1.0, this suggests a binding, and a compound wasconsidered a ‘hit’. Hits (RUactual to RUtheor of 0.7-2.0) were thenreviewed by a team of medicinal chemists and those with structuralpotentials were selected for further study. Selected molecules weretested in vitro in a solution coimmunoprecipitation assay usingrecombinant EWS-FLI1 and GST-RHA (647-1075).

General Method for the Synthesis and Analysis of Small MoleculeCompounds

Appropriated acetophenone (4.0 equv.) and catalytic amount ofdiethylamine (10 drops) were added to a solution of 4,7-dichloroisatin(1.0 equv.) in methanol (5 mL). The mixture was stirred at roomtemperature until starting material (4,7-dichloroisatin) disappearedcompletely. The resulted solution was concentrated and applied to flashchromatography eluting with Hexane/Ethyl acetate to afford pure productin quantitative yield. Further purification was done byrecrystallization with Hexane/Ethyl acetate. NMR spectra were recordedusing a Varian-400 spectrometer for ¹H (400 MHz), chemical shifts (8)are given in ppm downfield from tetramethylsilane as internal standard,and coupling constants (J-values) are in hertz (Hz). Elemental analyseswere performed by Atlantic Microlabs.

4,7-Dichloro-3-[2-(4-chlorophenyl)-2-oxoethyl]-3-hydroxy-1-1,3-dihydro-2H-indol-2-one(NSC635437): white solid; mp 194-196° C.; ¹H NMR (DMSO, 400 MHz) δ 10.96(s, 1H), 7.93 (d, 2H, J=8.8 Hz), 7.57 (d, 2H, J=8.8 Hz), 7.30 (d, 1H,J=8.8 Hz), 6.90 (d, 1H, J=8.8 Hz), 6.47 (s, 1H), 4.36 (d, 1H, J=18.4Hz), 3.71 (d, 1H, J=18.0 Hz). Anal. Calcd for C₁₆H₁₀Cl₃NO₃. 1/2 EtOAc:C, 52.09; H, 3.38; N, 3.38. Found: C, 52.53, H, 3.17, N, 3.55.

4,7-Dichloro-3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]-1,3-dihydro-2H-indol-2-one(YK-4-279): white solid; mp 149-151° C.; ¹H NMR (DMSO, 400 MH 1 z) δ10.93 (s, 1H), 7.86 (d, 2H, J=9.2 Hz), 7.26 (d, 1H, J=8.8 Hz), 6.98 (d,2H, J=8.8 Hz), 6.86 (d, 1H, J=8.4 Hz), 6.39 (s, 1H), 4.31 (d, 1H, J=18.0Hz), 3.80 (s, 3H), 3.61 (d, 1H, J=18.0 Hz). Anal. Calcd forC₁₇H₁₃Cl₂NO₄: C, 55.76; H, 3.58; N, 3.82. Found: C, 55.82, H, 3.98, N,3.51.

Fluorescence Polarization Assay

Increasing concentrations of FITC-E9R were added to a fixedconcentration of EWS-FLI1 (4.8 μM) to obtain a saturated binding curve.The assay was performed in 20 mM Tris, 500 mM NaCl, 0.67 M imidazole, pH7.4. The fluorescence polarization was analyzed in a QuantaMasterfluorimeter (Photon Technology International, Ford, West Sussex, UK)equipped with polymer sheet polarizers at an excitation wavelength of495 nm and emission wavelength of 517 nm. Increasing concentrations ofYK-4-279 were added to a fixed concentration of EWS-FLI1 and FITC-E9R(3.2 μM, as determined from saturated binding curve) with the samebuffer and instrumental settings as described above.

Plasmids and Reporter Assay

EGFP-E9R fusion constructs prepared as published (Frangioni, J. V. &Neel, B. G. Use of a general purpose mammalian expression vector forstudying intracellular protein targeting: identification of criticalresidues in the nuclear lamin A/C nuclear localization signal. J CellSci 105 (Pt 2), 481-488 (1993)). We transiently transfect the NR0B131luciferase reporter and full-length EWS-FLI1 into COS-7 cells withFugene-6 (Roche) and luciferase assay performed per manufacturer'sprotocol (Dual Luciferase Kit, Promega). Six hours followingtransfection, cells were treated with either 3 or 10 μM YK-4-279. Celllysates luciferase activity levels were standardized to renilla activityfrom a non-affected promoter and plotted as relative luciferase activity(RLA).

Caspase-3 Activity Measurement and Nuclear Fragmentation

Cells were treated for 24 hours with 10 μM YK-4-279. The Caspase-3substrate DEVD-AMC was incubated with equal amounts of protein lysateand fluorescence from cleaved substrate measured in a fluorimeter. TC32and non-transformed cells HEK-293, HFK, and HEC were treated for 6 hourswith high dose (50 μM) YK-4-279. DAPI stained cells were photographed at600× using inverted fluorescence microscope.

Mouse Strains and In Vivo Small Molecule Testing

One million TC71 or CHP-100 cells in 100 μL HBSS were injectedorthotopically into the gastrocnemius muscle of 4-8 week old SCID/bgmice (Taconic, Germantown, N.Y.). Prostate cancer xenografts wereestablished by subcutaneous injection of 5 million PC3 cells into theflanks of 4-8 week old nude mice (Taconic). Mice were randomized toreceive three times per week intraperitoneal injections of DMSO,YK-4-279 at 1.5 mg/dose when tumors were palpable. Each of the animalexperiments was begun with 10 mice that were randomized into treatmentand control groups when the tumors reached palpable size. In the controlgroups some tumors exceeded the IACUC maximal size (2 cm in anydimension) and were euthanized prior to day 14 and thus not included inthe day 14 analysis (FIG. 6 c). Tumor length and width were measuredevery 2-4 days and volume was calculated using the formula v=D×d2×n/6where D is the longest diameter and d is the shorter diameter. Xenograftstudies were approved by the Memorial Sloan-Kettering Cancer CenterInstitutional Animal Care and Use Committee.

RNAi for RHA and EWS-FLI1

shRNA (short hairpin RNA) against RHA was purchased from Open Biosystems(Huntsville, Ala.) Both virus production and purification were doneaccording to Open Biosystems protocols. (For designing shRNA seeMcIntyre G. J. et al. “Design and cloning strategies for constructingshRNA expression vectors.” BMC Biotechnol. (2006) 6:1)

shRNA RHA (nucleotides 598-618) #5 sequence (SEQ ID NO: 37):CCGGGAAGGATTACTACTCAAGAAACTCGAGTTTCTTGAGTAGT AATCCTTCTTTTT shRNA RHA(nucleotides 1689-1669) #7 sequence (SEQ ID NO: 38):CCGGTCGAGGAATCAGTCATGTAATCTCGAGATTACATGACTG ATTCCTCGATTTTT

Lentivirus infected cells were lysed after 6 days of selection withpuromycin.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism, available fromGraphPad Software Inc. of La Jolla, Calif.

Results RHA is a Validated Target in ESFT

A region of RHA that binds to EWS-FLI1 was identified based uponphage-display epitope screening (Toretsky, J. A., et al. OncoproteinEWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 66,5574-5581 (2006)) (FIG. 29A). To validate RHA as critical in ESFT cells,RHA levels were reduced using shRNA and ESFT cell viability was reducedby 90% (FIGS. 29B, 29C). A pancreatic cell line, PANC1 cells that do notexpress EWS-FLI1, were stably transfected with the same shRNA vectorswith similar reduction in RHA levels (FIG. 35), but with no decrease incell viability (FIG. 35). In order to further validate theprotein-protein interaction of RHA with EWS-FLI1 as a therapeutic targetfor ESFT patients, site-directed mutagenesis was performed on theGST-RHA (647-1075) protein fragment. GST-RHA (647-1075) mutants wereexpressed and coimmunoprecipitated with full length recombinantEWS-FLI1. Mutants P824A and D827A showed a significant decrease inbinding compared to wild-type control (FIG. 29D). The full length RHAmutant D827A maintained wild-type ATPase activity (FIG. 36); therefore,the D827A mutant was chosen to test whether RHA binding to EWS-FLI1 wasrequired for neoplastic transformation.

RHA is Required for EWS-FLI1 Modulated Transformation

Murine embryonic fibroblasts (W) that express low levels of endogenousRHA were stably transfected with EWS-FLI1 (WEF1) and either full-lengthwild-type RHA or full-length RHA (D827A). A greater than additive effectwas observed when comparing the colony numbers from W+RHA (227±66) andWEF1 (115±8) to those of WEF1+RHA (582±30) (FIGS. 29E, 29F). The RHA(D827A) expressing cells demonstrated 3-fold lower anchorage-independentgrowth (p=0.0028) than the wild-type (FIGS. 29E, 29F). Similar proteinexpression levels of EWS-FLI1 and RHA were obtained in the fibroblasts(FIG. 29G). The EWS-FLI1 immunoblot was evaluated by densitometry anddemonstrated reasonably similar protein levels amongst derived cellpopulations (FIG. 29H). The significant reduction of colony formation bythe RHA (D827A) expressing cells suggests a critical role inanchorage-independent growth that is abrogated by RHA not binding toEWS-FLI1.

E9R Peptide Blocks RHA Binding to EWS-FLI1

Reagents were developed to block RHA binding to EWS-FLI1 since RHA isnecessary for optimal EWS-FLI1 activity. The E9R peptide corresponds toamino acids 823 to 832, located in the proximal HA2 region of RHA (FIG.29A). A cell-free protein interaction assay was developed to testwhether E9R peptide inhibits RHA binding to EWS-FLI1. Thisimmunoprecipitation assay demonstrated binding between bacteriallyexpressed GST-RHA (647-1075) and full-length purified recombinantEWS-FLI1 (FIG. 30A, lane 3). Titration of E9R demonstrated adose-dependent reduction in the binding of GST-RHA (647-1075) andfull-length EWS-FLI1 with a decreased association to 50% with 0.1 μM E9R(FIG. 30A, lane 6). It was determined whether disrupted EWS-FLI1/RHAbinding inhibited cell growth.

E9R Peptide Specifically Inhibits ESFT Growth

Peptide delivery to growing cells is greatly facilitated by cellpermeable peptides (CPP) 27. Antennapedia (Antp) is a CPP that wassynthesized on the amino-terminus of E9R with or without the D827Amutation (E9R-P and E9R(D5A)-P, respectively) (Table 5).

TABLE 5 SEQ ID Name Amino Acid Sequence NO Antp: Antennapedia-RQIKIWFQNRRMKWKK 34 E9R-P: [FITC]- 35 Antennnapedia-E9RRQIKIWFQNRRMKWKKPPPLDAVIEA D5A: Antennapedia- [FITC]- 36 E9R-RQIKIWFQNRRMKWKKPPPLAAVIEA D5A [FITC] conjugated fluorescent dye

Monolayer cultures of the EWS-FLI1-positive ESFT cell line, TC32, or acontrol EWS-FLI1-negative cell line, SKNAS (neuroblastoma), were treatedwith fluorescein conjugated peptides. Only the EWS-FLI1 containing cellsshowed reduced growth (FIG. 30B) and the SKNAS cells showed mildstimulation from the peptide based on an unknown mechanism. Confocalmicroscopy demonstrated uptake throughout the cell, including nuclei(shown by DAPI overlay, FIG. 30C). E9R-P significantly reduced ESFT cellgrowth (p=0.048) while neither the D5A mutated control nor antennapediapeptides alone reduced ESFT cell growth (FIG. 30D). Neuroblastoma cellstreated with the same peptides did not have a statistically significantalteration in growth, although a slight increase was observed withE9R(D5A) treated cells (p=0.175). To determine the effect of E9R uponanchorage-independent growth, we stably transfected E9R as an EGFPfusion protein into TC71 (ESFT) or SKNAS (neuroblastoma) cells. Anin-frame expression of LQLPPLERLTL excluded E9R from the nucleus28.Transfected cells showed E9R peptide expression either throughout thecell or excluded from the nucleus as predicted based on the intendedtargeting (FIG. 30E). TC71 colony formation was significantly reduced by95% due to the expression of E9R, except when the peptide was excludedfrom the nucleus (FIGS. 30E, 30F). The anchorage-independent growth ofSKNAS was not affected by the E9R peptide (FIGS. 30E, 30F). To furthersupport specificity of the E9R peptide, a second small round blue celltumor, embryonal rhabdomyosarcoma (RD cells), expressing pGE9R did notshow reduced anchorage-independent growth.

Small Molecule Binds to EWS-FLI1

A library of 3000 small molecules (NCI, DTP) was screened for EWS-FLI1binding using surface plasmon resonance (SPR). Compounds were selectedthat exhibited a binding level (actual resonance units, RUactual) toRUtheor ratio between 0.7 and 2.0 suggesting monomeric binding toEWS-FLI1 and also had favorable drug-like properties (Leeson, P. D. &Springthorpe, B. The influence of drug-like concepts on decision-makingin medicinal chemistry. Nature reviews 6, 881-890 (2007)). NSC635437 hadan RUactual:RUtheor of 0.9 and was chosen for further evaluation basedupon potential for chemical derivatization. 1.0 gram of NSC635437 wassynthesized to complete the studies and for use as a standard (FIG.31A).

Methoxy-Derivative is a More Potent Inhibitor of EWS-FLI1 Binding to RHA

In a cell-free assay, NSC635437 reduced the direct binding of GST-RHA(647-1075) to full-length recombinant EWS-FLI1 (FIG. 31B, left panel).An aromatic optimization strategy was used to design analogs to improveupon the activity of NSC635437. One of these compounds (YK-4-279),substituted with a methoxy group at the para position (p-methoxy) of thearomatic ring (FIG. 31A) significantly reduced the protein-proteininteraction of EWS-FLI1 with GST-RHA (647-1075) in vitro (FIG. 31B,right panel). A KD of 9.48 μM was calculated for the affinity ofYK-4-279 with EWS-FLI1 using surface plasmon resonance (FIG. 31C). Tosupport a model of YK-4-279 as having similar interaction qualities toE9R, SPR displacement assay shows 10 μM YK-4-279 reducing the binding of64 μM E9R from 17 R.U. to 7 R.U. and 32 μM E9R from 13 R.U. to 5 R.U.(FIG. 31D). Fluorescence polarization further demonstrated E9Rdisplacement of E9R when YK-4-279 was titrated into the experiment,showing complete displacement at 30 μM YK-4-279 (FIG. 31E).

YK-4-279 Demonstrates Functional Inhibition of EWS-FLI1

ESFT cells treated with YK-4-279 demonstrated a dissociation of EWS-FLI1from RHA by 10 μM, consistent with the KD value (FIG. 32A, top panel).YK-4-279 did not directly affect EWSFLI1 nor RHA levels (FIG. 32A,middle and lower panels and FIG. 37). To further support YK-4-279 as afunctional inhibitor of EWS-FLI1, COS7 cells were cotransfected withEWS-FLI1 and NROB1 reporter-luciferase plasmid (containing EWS-FLI1regulatory GGAA elements (Gangwal, K., et al. Microsatellites as EWS/FLIresponse elements in Ewing's sarcoma. Proc Natl Acad Sci USA 105,10149-10154 (2008)). The EWS-FLI1 transfected cells demonstrated adose-dependent decrease in promoter activity when treated for 18 hourswith 3 and 10 μM YK-4-279 (FIG. 32B). As an additional control fornon-specific promoter effects, an NFκB responsive reporter wastransfected into COS7 cells and activated with PMA. YK-4-279 did notaffect the NFκB responsive promoter (FIG. 38A). In a recent publication,EWS-FLI1 was shown to modulate cyclin D protein levels by altering acyclin D splice site (Sanchez, G., et al. Alteration of cyclin D1transcript elongation by a mutated transcription factor up-regulates theoncogenic D1b splice isoform in cancer. Proc Natl Acad Sci USA (2008)).Blocking the interaction of EWS-FLI1 with RHA using YK-4-279 nearlyeliminated cyclin D levels in TC32 cells treated for 14 hours (FIG.32D), but did not affect cyclin D levels in four non-EWS-FLI1 containingcell lines (FIG. 38B, FIG. 38C).

YK-4-279 Specifically Inhibits ESFT Cell Growth and Induces Apoptosis

The compound identified from screening, NSC635437, was found to have anIC50 of 20 μM for TC32 cells growing in monolayer; however, YK-4-279reduced the IC50 to 900 nM (FIG. 33A).

YK-4-279 was relatively specific for ESFT cells as compared to thenon-transformed HEK293 cells, demonstrating a 10-fold difference in IC50(FIG. 33B). Primary cell lines, ES925 and GUES1, established from ESFTpatients with recurrent tumors demonstrated sensitivity to YK-4-279 withanti-proliferative IC50 values of 1 and 8 μM, respectively (FIG. 33C). Apanel of ESFT cell lines demonstrated IC50 values between 0.5 to 2 μMfor YK-4-279 while cell lines that lack EWSFLI1 have IC50 values inexcess of 25 μM (FIG. 33D). An additional panel of non-transformedkeratinocytes (HFK) and ectocervical cells (HEC) treated for 3 days with30 μM YK-4-279 showed an IC50 that exceeded 30 μM (FIG. 39A).

Apoptosis leads to tumor cell death through the activation of sequentialcaspase enzymes, with caspase-3 demonstrating a commitment to cellularsuicide (Li, F., et al. Control of apoptosis and mitotic spindlecheckpoint by survivin. Nature 396, 580-584 (1998). Caspase-3 activityrose in a dose dependent fashion in TC32 cells treated with YK-4-279 for24 hours (FIG. 39B).

Caspase activity was similar to 1 μM doxorubicin, a standard agent inthe treatment of patients with ESFT6. Additional malignant andnon-malignant cell lines were evaluated for caspase-3 activation inresponse to YK-4-279. While YK-4-279 induced caspase-3 activity in fourESFT cell lines (TC32, A4573, TC71, and ES925), none of the 5non-EWS-FLI1 cancer cell lines nor 3 non-transformed cell lines (HFK,HEC, HEK293) treated with YK-4-279 resulted in apoptosis (FIG. 33E).Treatment of TC32, HEK293, HFK, and HEC with short-term (6 hours) highdose (50 μM) YK-4-279 resulted in significant apoptosis of the ESFTcells but no cell death in the nontransformed cells (FIG. 33F).Together, these results support the specific toxicity of YK-4-279 uponcell lines containing EWS-FLI1 compared with other tumor andnon-transformed cells.

In order to further support for the target specificity of YK-4-279toxicity upon ESFT cells, the levels of each of the critical proteinswere reduced by using shRNA in A673 cells42. The RHA reduced cellsdemonstrated a YK-4-279 IC50 of >10 μM, while control shRNA (targetingluciferase) IC50 was less than 1 μM (FIG. 39C). When EWS-FLI1 wasreduced using shRNA, the IC50 increased 10-fold from 0.5 μM toapproximately 5 μM (FIG. 39D, FIG. 39E).

ESFT Xenograft Growth is Inhibited by YK-4-279

ESFT (orthotopic) or prostate cancer cell xenograft tumors wereestablished in SCID/bg mice. Tumor growth rate was reduced for CHP-100,ESFT, (FIG. 6A), but not the PC3, prostate tumors (FIG. 34B). Fiveindependent experiments were performed with the ESFT xenografts (TC71and CHP-100) and the cumulative data for these experiments shows amarked overall tumor reduction (p<0.0001) in the YK-4-279 treatedanimals (FIG. 34C). Pathological analysis of animals treated withYK-4-279 did not show any signs of toxicity except changes related to IPinjection. Tumors from animals treated with YK-4-279 were compared withDMSO treatment using immunohistochemistry to identify caspase-3 activity(FIG. 34D). The CHP-100 xenograft tumors from treated animals had a3-fold increase in caspase-3 activity compared to control animals (FIG.34E). These results show inhibition of two ESFT tumor models andconcomitant increased apoptosis following YK-4-279 treatment.

There is a significant need for new cancer therapies that enhanceefficacy and reduce long-term morbidity. Protein products oftumor-specific chromosomal translocations, which are present only incancer cells, provide unique targets for anti-tumor therapies1. Thesetranslocations span a broad range of malignancies, including carcinomas,hematopoietic malignancies, and sarcomas (French, C. A., et al. Midlinecarcinoma of children and young adults with NUT rearrangement. J ClinOncol 22, 4135-4139 (2004); Helman, L. J. & Meltzer, P. Mechanisms ofsarcoma development. Nat Rev Cancer 3, 685-694 (2003); Poppe, B., et al.Expression analyses identify MLL as a prominent target of 11q23amplification and support an etiologic role for MLL gain of function inmyeloid malignancies. Blood 103, 229-235 (2004)). In many cancers, thesetranslocations lead to novel fusion proteins that both initiate andmaintain oncogenesis. While some of these translocations, such asBCR-ABL5, lead to constitutively activated kinases, the majority lead tofusion proteins that function as transcription factors and lackintrinsic enzymatic activity. These translocation-generatedtranscription factor fusion proteins are ideal targets of anti-cancertherapies, yet no pharmaceuticals have been developed towards thesetargets.

The Ewing's sarcoma family of tumors (ESFT) can occur anywhere in thebody and most often in the 2nd and 3rd decades. ESFT often respond wellto initial chemotherapy, yet 40% of patients will develop recurrentdisease. The majority of patients with recurrent disease will die fromESFT, while 75-80% of patients who present with metastatic ESFT will diewithin 5 years despite high-dose chemotherapy (Grier, H. E., et al.Addition of ifosfamide and etoposide to standard chemotherapy forEwing's sarcoma and primitive neuroectodermal tumor of bone. N Engl JMed 348, 694-701 (2003)). ESFT contain a well-characterized chromosomaltranslocation that fuses the amino-half of EWS to the carboxy-half of anets-family DNA binding protein (Delattre, O., et al. The Ewing family oftumors—a subgroup of small-round-cell tumors defined by specificchimeric transcripts. N Engl J Med 331, 294-299 (1994)). The most commonfusion protein is the oncogenic transcription factor EWS-FLI1.Elimination of EWS-FLI1 using antisense and siRNA approaches results inthe prolonged survival of ESFT xenograft-bearing animals (Hu-Lieskovan,S., Heidel, J. D., Bartlett, D. W., Davis, M. E. & Triche, T. J.Sequencespecific knockdown of EWS-FLI1 by targeted, nonviral delivery ofsmall interfering RNA inhibits tumor growth in a murine model ofmetastatic Ewing's sarcoma. Cancer Res 65, 8984-8992 (2005)), but thisapproach currently lacks translation to clinical therapy (Kovar, H.,Ban, J. & Pospisilova, S. Potentials for RNAi in sarcoma research andtherapy: Ewing's sarcoma as a model. Semin Cancer Biol 13, 275-281(2003); Tanaka, K., Iwakuma, T., Harimaya, K., Sato, H. & Iwamoto, Y.EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of humanEwing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest99, 239-247 (1997)). Small-molecule targeting would be directed towardsthe disruption of EWS-FLI1 from established transcriptional complexes,since EWS-FLI1 lacks intrinsic enzymatic activity. The EWS-FLI1transcriptional complex includes: RNA polymerase II, CREB-bindingprotein (CBP), and RNA Helicase A (RHA) (Petermann, R., et al. OncogenicEWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II.Oncogene 17, 603-610 (1998); Nakatani, F., et al. Identification ofp21WAF1/CIP1 as a direct target of EWS-Fli1 oncogenic fusion protein. JBiol Chem 278, 15105-15115 (2003); Toretsky, J. A., et al. OncoproteinEWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 66,5574-5581 (2006); Zhong, X. & Safa, A. R. RNA helicase A in the MEF1transcription factor complex upregulates the MDR1 gene inmultidrug-resistant cancer cells. J Biol Chem 279, 17134-17141 (2004);Nakajima, T., et al. RNA helicase A mediates association of CBP with RNApolymerase II. Cell 90, 1107-1112 (1997)). Previous investigationsshowed RHA augments EWSFLI1 modulated oncogenesis, suggesting that thisprotein-protein complex is particularly important for tumor maintenance(Toretsky, J. A., et al. Oncoprotein EWS-FLI1 activity is enhanced byRNA helicase A. Cancer Res 66, 5574-5581 (2006)). Small moleculeinhibitors that block RHA interaction by targeting the oncogenic fusionprotein EWS-FLI1 are the first in a new class of anti-tumor therapy. RHAhas a critical role in embryogenesis and thus is a reasonable partnerfor an oncogene in a highly undifferentiated tumor. RHA is indispensablefor ectoderm survival in gastrulation of mammals (Lee, C. G., et al. RNAhelicase A is essential for normal gastrulation. Proc Natl Acad Sci USA95, 13709-13713 (1998)) and is required beyond embryogenesis because RHAnull mouse fibroblast cells are not viable. However, transient reductionof RHA protein levels in COS cells did not affect the viability(Hartman, T. R., et al. RNA helicase A is necessary for translation ofselected messenger RNAs. Nat Struct Mol Biol (2006)). RHA provides atranscriptional coactivator role in models of tumorigenesis includingNFkappaB and STATE transcriptomes. RHA binds to DNA in a sequencespecific manner upon the promoters of p16^(INK)4a and MDR1 (Tetsuka, T.,et al. RNA helicase A interacts with nuclear factor kappaB p65 andfunctions as a transcriptional coactivator. Eur J Biochem 271, 3741-3751(2004); Valineva, T., Yang, J. & Silvennoinen, O. Characterization ofRNA helicase A as component of STATE-dependent enhanceosome. NucleicAcids Res 34, 3938-3946 (2006); Myohanen, S. & Baylin, S. B.Sequence-specific DNA binding activity of RNA helicase A to the p16INK4a promoter. J Biol Chem 276, 1634-1642 (2001); Zhong, X. & Safa,A. R. RNA helicase A in the MEF1 transcription factor complexupregulates the MDR1 gene in multidrug-resistant cancer cells. J BiolChem 279, 17134-17141 (2004); Nakajima, T., et al. RNA helicase Amediates association of CBP with RNA polymerase II. Cell 90, 1107-1112(1997)). The amino-terminal region of RHA is most often the site forprotein-protein interactions. CBP binds aa 1-250 of RHA 20, whileadditional partners for RHA bind in the amino-terminal region includingRNA polymerase II21 and BRCA121. In modulating RNA interference in theRISC complex, RHA and Dicer, TRBP and Ago2 interact in the region of aa1-272 of RHA22. EWS-FLI1 binds to RHA in a unique region that is notoccupied by other transcriptional nor RNA metabolism proteins (Toretsky,J. A., et al. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicaseA. Cancer Res 66, 5574-5581 (2006)), thus increasing the attractivenessof this protein target.

Disruption of protein-protein interactions by small molecules is arapidly evolving field. Proteins with more flexible structures, in somecases disordered proteins, have a greater potential for small moleculebinding than rigid proteins because of higher induced fit samplingprobabilities (Bhalla, J., Storchan, G. B., MacCarthy, C. M., Uversky,V. N. & Tcherkasskaya, O. Local flexibility in molecular functionparadigm. Mol Cell Proteomics 5, 1212-1223 (2006)). A disordered proteinis defined, in part, by increased intrinsic movement and the inabilityto form rigid 3-dimensional structure (Xie, H., et al. Functionalanthology of intrinsic disorder. 1. Biological processes and functionsof proteins with long disordered regions. Journal of proteome research6, 1882-1898 (2007)). EWS-FLI1 is a disordered protein and requires thedisorder for maximal transactivation of transcription (Ng, K. P., et al.Multiple aromatic side chains within a disordered structure are criticalfor transcription and transforming activity of EWS family oncoproteins.Proc Natl Acad Sci USA 104, 479-484 (2007); Uren, A., Tcherkasskaya, O.& Toretsky, J. A. Recombinant EWS-FLI1 oncoprotein activatestranscription. Biochemistry 43, 13579-13589 (2004)). Based on theseobservations, EWS-FLI1, along with its binding to RHA, can provide aunique drug target.

EWS-FLI1 is a unique, cancer specific, molecule that can have utility asa therapeutic target in ESFT cells. RHA is critical to the function ofEWS-FLI1. A peptide (E9R) that blocks RHA binding to EWS-FLI1specifically reduced anchorage-independent growth. Use of the smallmolecule lead compound, NSC635437, that binds to EWS-FLI1, and the leadcompound derivative YK-4-279, along with E9R peptide, demonstrate thatthe EWS-FLI1/RHA interaction can be blocked with a detrimental effect onESFT cells both in vitro and in vivo. These findings validate a highlyspecific cancer target, the interaction of EWS-FLI1 with RHA.

It was demonstrated that the small molecule YK-4-279 binds to EWS-FLI1and blocks the binding of RHA. A series of xenograft experimentsdemonstrated that 60-75 mg/kg YK-4-279 significantly reduced tumorgrowth. The small molecule not only inhibited RHA binding to EWS-FLI1,but also reduced EWS-FLI1 modulated transcription. An additionalputative function of EWS-FLI1 is splice-site modification (Knoop, L. L.& Baker, S. J. EWS/FLI alters 5′-splice site selection. J Biol Chem 276,22317-22322. (2001)), which was recently supported by the EWS-FLI1altered splicing of cyclin D132. Treatment of ESFT cells with YK-4-279led to decreased cyclin D1 levels. Additional investigations of thesplicing complex are necessary to determine if this effect is due to thedisruption of an EWS-FLI1/RHA complex or allosteric interference withEWS-FLI1. Small molecule inhibitors have use as therapeutics, and arealso useful as functional probes.

EWS-FLI1 was recognized as a potential therapeutic target over 15 yearsago, almost immediately after the protein was identified as a product ofthe breakpoint region t(11; 22) 35. The disordered biophysical nature ofEWS-FLI1 precludes standard structure-based small molecule design (Uren,A., Tcherkasskaya, O. & Toretsky, J. A. Recombinant EWS-FLI1 oncoproteinactivates transcription. Biochemistry 43, 13579-13589 (2004).).Therefore, development of small molecule protein-protein inhibitors waspursued based upon the assumption that EWS-FLI1 would have a bindingpartner critical for its oncogenic function, which we previouslyidentified as RHA13, and validated in the current study. The exactnature of the requirement of RHA for EWS-FLI1 is currently underinvestigation, however it is believed that RHA could be involved inEWS-FLI1 function, synthesis or stability. These data support multiplemechanisms and therefore require further enzymatic and structuralstudies of EWS-FLI1 bound RHA for resolution. The fact that YK-4-279 isstill toxic to A673 cells with reduced EWS-FLI1 could be due to residualEWS-FLI1 or suggest broader action of the compound. In addition, whiledata suggest that YK-4-279 is specific for ESFT cell toxicity, YK-4-279may reveal other protein interactions.

Inhibitory peptides offer a greater likelihood of specificity tovalidate protein-protein interaction targets and to evaluateprotein-complex disruption; however, peptides are problematic forclinical development. Peptides were used to compare the effects ofdisrupting protein-protein interactions with our small molecules. Whilesmall peptides are currently being developed as therapeutic agents(Plescia, J., et al. Rational design of shepherdin, a novel anticanceragent. Cancer Cell 7, 457-468 (2005); Palermo, C. M., Bennett, C. A.,Winters, A. C. & Hemenway, C. S. The AF4-mimetic peptide, PFWT, inducesnecrotic cell death in MV4-11 leukemia cells. Leuk Res (2007)), 10-20 aapeptides present formidable pharmacokinetic stability and deliverychallenges. The E9R peptide may compete with full-length RHA binding toEWS-FLI1 and data support a functional displacement of RHA by E9R. Usingsurface plasmon resonance and fluorescence polarization, it wasdemonstrated that YK-4-279 can ‘displace’ E9R from EWS-FLI1. While theseresults support E9R and YK-4-279 binding to the same site on EWS-FLI1,allosteric interference cannot be excluded. Therefore, a structuralmodel of EWSFLI1 is required to both fully prove this interaction andYK-4-279 binding site, but is yet unavailable due to the challenges ofdisordered proteins (Bhalla, J., Storchan, G. B., MacCarthy, C. M.,Uversky, V. N. & Tcherkasskaya, O. Local flexibility in molecularfunction paradigm. Mol Cell Proteomics 5, 1212-1223 (2006)).

The interaction of RHA with EWS-FLI1 presents an ideal opportunity forthe development of small molecule protein-protein interaction inhibitors(SMPPII). Both evidence and prevailing opinion support disorderedproteins as potential targets of small molecule therapeutics (Cheng, Y.,et al. Rational drug design via intrinsically disordered protein. TrendsBiotechnol 24, 435-442 (2006).). This data also support EWS-FLI1 proteininteraction targeting to modulate oncogene function and potentially leadto novel therapeutics. Small molecules that disable EWS-FLI1 functionwith minimal toxicity, in particular sparing of hematopoetic stem cells,can potentially provide a valuable adjuvant therapy for patients withESFT. In addition, this paradigm for drug discovery can be applied tomany related sarcomas that share similar oncogenic fusion proteins.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention.

1. A compound having a formula:

or salt thereof.
 2. The compound of claim 1, wherein the salt is a pharmaceutically acceptable salt.
 3. A method for treating cancer comprising administering an effective amount of a compound having a formula:

or pharmaceutically acceptable salt thereof to a subject in need thereof.
 4. The method of claim 3, wherein the subject is a mammal.
 5. The method of claim 4, wherein the mammal is a human.
 6. The method of claim 3, wherein the cancer is selected from the group consisting of Ewing's sarcoma, clear-cell sarcoma, myxoid liposarcoma, desmoplastic small round-cell tumor, myxoid chondrosarcoma, myxoid liposarcoma, acute myeloid leukemia, congenital fibrosarcoma, prostate cancer, pancreatic cancer, alveolar rhabdomyosarcoma, synovial sarcoma, dermatofibrosarcoma protuberans, inflammatory myofibroblastic tumor, and alveolar soft-part sarcoma.
 7. The method of claim 3, wherein the cancer is Ewing's sarcoma.
 8. The method of claim 3, wherein the subject exhibits a gene translocation.
 9. The method of claim 8, wherein the gene translocation encodes a fusion gene selected from the group consisting of EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4, EWSR1-FEV, EWSR1-ATF1, EWSR1-WT1, EWSR1-NR4A3, FUS-DDIT3, EWSR1-DDIT3, PAX3-FOXO1A, PAX7-FOXO1A, SYT-SSX, COL1A1-PDGFB, ETV6-NTRK3, TMP3-ALK, TMP4-ALK, and ASPL-TFE3.
 10. The method of claim 3, further comprising administering an additional therapeutic agent.
 11. The method of claim 10, wherein the additional therapeutic agent is selected from the group consisting of vinca alkaloid, anthracycline, anthracene, epipodophyllo-toxin, actinomyocin D, mithomycin C, mitramycin, methotrexate, docetaxel, etoposide (VP-16), paclitaxel, docetaxel, and adriamycin.
 12. A pharmaceutical composition comprising a compound having a formula:

or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or diluent. 